Adzymes and uses thereof

ABSTRACT

Disclosed is a family of novel protein constructs, useful as drugs and for other purposes, termed “adzymes,” comprising an address moiety and a catalytic domain. In some types of disclosed adzymes, the address binds with a binding site on or in functional proximity to a targeted biomolecule, e.g., an extracellular targeted biomolecule, and is disposed adjacent the catalytic domain so that its affinity serves to confer a new specificity to the catalytic domain by increasing the effective local concentration of the target in the vicinity of the catalytic domain. The present invention also provides pharmaceutical compositions comprising these adzymes, methods of making adzymes, DNA&#39;s encoding adzymes or parts thereof, and methods of using adzymes, such as for treating human subjects suffering from a disease, such as a disease associated with a soluble or membrane bound molecule, e.g., an allergic or inflammatory disease.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional PatentApplication Serial No. 60/406,517, filed Aug. 27, 2002, U.S. ProvisionalPatent Application Serial No. 60/423,754, filed Nov. 5, 2002, and U.S.Provisional Patent Application Serial No. 60/430,001, filed Nov. 27,2002, the entire contents of each of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0002] This invention relates to synthetic protein constructs useful inmodulating a variety of targeted molecules in situ. In particularaspects, it relates to a family of constructs employing linked molecularparts which target and modulate the activity of a biomoleculecatalytically to induce a therapeutic effect.

[0003] Many diseases are caused by or associated with biomolecules,either free in solution in body fluids or exposed to extracellular bodyfluids such as membrane-bound proteins and polysaccharides, such ascytokines or growth factors, and it is widely recognized that it ispossible to develop therapies for such diseases by modulating theactivity of the biomolecule.

[0004] For example, overproduction of TNF-α and/or TNF-β is closelylinked to the development of many diseases, including septic shock,adult respiratory distress syndrome, rheumatoid arthritis, selectiveautoimmune disorders, graft-host disease following bone marrowtransplantation and cachexia. Other diseases associated with excessiveTNF-α and/or TNF-β production include hemorrhagic shock, asthma andpost-renal dialysis syndrome. The multiplicity of actions of TNF-α andTNF-β can be ascribed to the fact that TNF-α and/or TNF-β actions resultin activation of multiple signal transduction pathways, kinases,transcription factors, as well as an unusually large array of cellulargenes. (Walajtys-Rode, Elizbieta, Kosmos (Warsaw), 44, 451-464, 1995,C.A. 124:199735a, 1995). TNFα has also been linked to the development ofautoimmune disorders.

[0005] Current therapies for combating the foregoing disorders includethe administration of a binding agent, such as an antibody or solublereceptor, that binds to and thereby inhibits a targeted biomolecule thatcauses or is associated with the disease. However, there are manydrawbacks associated with this approach. For example, binding agents, bytheir very nature, can only inhibit the biomolecules(s) to which theyare bound, and can neither catalytically inactivate a series ofbiomolecules nor chemically alter the bound biomolecules(s). It isprobably for these reasons that relatively large doses of binding agentsare often needed to achieve therapeutic effectiveness, exposing thesubject to dangerous and often toxic side-effects. Moreover, productionof such large quantities of antibodies and other binding agents isexpensive.

[0006] Targeted therapeutic agents with greater effectiveness thantraditional binding agent therapeutics would be a desirable improvement.

SUMMARY OF THE INVENTION

[0007] In certain aspects, the invention provides a new class ofengineered protein constructs, referred to herein as “adzymes”, as wellas methods and compositions related to the use and production ofadzymes. Adzymes are chimeric protein constructs that join one or morecatalytic domains with one or more targeting moieties (or “addresses”).A catalytic domain of an adzyme has an enzymatically active site thatcatalyzes a reaction converting a pre-selected substrate (the “target”or “targeted substrate”) into one or more products, such as by cleavage,chemical modifications (transformations) or isomerization. Such productsmay have an altered activity relative to the substrate, optionallyhaving an increased or decreased activity or an activity that isqualitatively different.

[0008] In certain aspects, the invention provides adzymes comprising acatalytic domain and a targeting moiety, wherein the catalytic domaincatalyzes a chemical reaction converting a substrate into one or moreproducts, and wherein the targeting moiety reversibly binds to anaddress site that is either on the substrate or in functional proximitywith the substrate. Preferably, the targeting moiety binds reversibly tothe address site. Optionally, said targeting moiety and said catalyticdomain are heterologous with respect to each other. Generally, saidtargeting moiety, when provided separately, binds to the substrate, andsaid catalytic domain, when provided separately, catalyzes the chemicalreaction converting said substrate to one or more products.

[0009] In certain embodiments, a catalytic domain and a targeting domainof the adzyme are joined by a polypeptide linker to form a fusionprotein. A fusion protein may be generated in a variety of ways,including chemical coupling and cotranslation. In a preferredembodiment, the fusion protein is a cotranslational fusion proteinencoded by a recombinant nucleic acid. In certain embodiments the linkerfor the fusion protein is an unstructured peptide. Optionally, thelinker includes one or more repeats of Ser₄Gly or SerGly₄. In preferredembodiments, the linker is selected to provide steric geometry betweensaid catalytic domain and said targeting moiety such that said adzyme ismore effective against the substrate than either the catalytic domain ortargeting moiety alone. For example, the linker may be selected suchthat the adzyme is more potent than said catalytic domain or targetingmoiety with respect to the reaction with said substrate. The linker maybe selected such that the targeting moiety presents the substrate to theenzymatic domain at an effective concentration at least 5 fold greaterthan would be present in the absence of the targeting moiety.

[0010] In certain embodiments, the adzyme is an immunoglobulin fusion,wherein the catalytic domain and the targeting moiety are joined, in ageometry consistent with effectiveness against substrate, to at least aportion of an immunoglobulin comprising a constant domain of animmunoglobulin. For example, the adzyme may comprise a first fusionprotein and a second fusion protein, wherein the first fusion proteincomprises a constant portion of an immunoglobulin heavy chain and acatalytic domain, and wherein the second fusion protein comprises aconstant portion of an immunoglobulin heavy chain and a targeting domainthat reversibly binds with an address site on or in functional proximityto the substrate. Preferably the immunoglobulin portions are Fc portionsthat dimerize by disulfide bonds.

[0011] In certain embodiments, an adzyme is designed so as to have oneor more desirable properties, with respect to the reaction with saidsubstrate. In many instances, such properties will be significant forachieving the desired effect of the adzyme on the substrate. Forexample, an adzyme may have a potency at least 2 times greater than thepotency of catalytic domain or the targeting moiety alone, andpreferably at least 3, 5, 10, 20 or more times greater than the potencyof the catalytic domain or targeting moiety alone. An adzyme may have ak_(on) of 10³ M⁻¹s⁻¹ or greater, and optionally a k_(on) of 10⁴ M⁻¹s⁻¹,10⁵ M⁻¹s⁻¹, 10⁶ M⁻¹s⁻¹, 10⁷ M⁻¹s⁻¹ or greater. An adzyme may have ak_(cat) of 0.1⁻⁴ sec⁻¹ or greater, and optionally a k_(cat) of 1 sec⁻¹,10 sec⁻¹, 50 sec⁻¹ or greater. An adzyme may have a K_(D) that is atleast 5, 10, 25, 50 or 100 or more fold less than the K_(m) of thecatalytic domain. An adzyme may have a k_(off) of 10⁻⁴ sec⁻¹ or greater,and optionally a k_(off) of 10⁻³ sec⁻¹, 10⁻² sec⁻¹, or greater. Anadzyme may have a catalytic efficiency that is at least 5 fold greaterthan the catalytic efficiency of the catalytic domain alone, andoptionally a catalytic efficiency that is at least 10 fold, 20 fold, 50fold or 100 fold greater than that of the catalytic domain. An adzymemay have a K_(m) at least 5 fold, 10 fold, 20 fold, 50 fold, or 100 foldless than the K_(m) of the catalytic domain alone. An adzyme may have aneffective substrate concentration that is at least 5 fold, 10 fold, 20fold, 50 fold or 100 fold greater than the actual substrateconcentration. In certain preferred embodiments, an adzyme will bedesigned so as to combine two or more of the above described properties.

[0012] A catalytic domain may include essentially any enzymatic domainthat achieves the desired effect on a selected substrate. The catalyticdomain may be selected so as to modify one or more pendant groups ofsaid substrate. The substrate may include a chiral atom, and saidcatalytic domain may alter the ratio of stereoisomers. The catalyticdomain may alter the level of post-translational modification of thepolypeptide substrate, such as a glycosylation, phosphorylation,sulfation, fatty acid modification, alkylation, prenylation oracylation. Examples of enzymatic domains that may be selected include: aprotease, an esterase, an amidase, a lactamase, a cellulase, an oxidase,an oxidoreductase, a reductase, a transferase, a hydrolase, anisomerase, a ligase, a lipase, a phospholipase, a phosphatase, a kinase,a sulfatase, a lysozyme, a glycosidase, a nuclease, an aldolase, aketolase, a lyase, a cyclase, a reverse transcriptase, a hyaluronidase,an amylase, a cerebrosidase and a chitinase. Regardless of the type ofcatalytic domain, it may be desirable that the adzyme be resistant toautocatalysis (e.g., inter- or intra-molecular reactions), particularlyat an adzyme concentration that is about equal to the concentration ofadzyme in a solution to be administered to a subject. In certainembodiment, the adzyme acts on the substrate such that a product of thechemical reaction is an antagonist of the substrate.

[0013] In certain preferred embodiments, the catalytic domain of anadzyme includes a protease domain that, when active, cleaves at leastone peptide bond of a polypeptide substrate. In general it will bedesirable to design the adzyme such that it is resistant to cleavage bythe protease catalytic domain. The protease domain may be generated as aaymogen (an inactive form) and then activated prior to use. The adzymemay be purified from a cell culture in the presence of a reversibleprotease inhibitor, and such inhibitor may be included in any subsequentprocessing or storage activities.

[0014] A targeting moiety may include essentially any molecule orassembly of molecules that binds to the address site (e.g., on thesubstrate in the case of direct adzymes or on a molecule that occurs infunctional proximity to the substrate, in the case of proximityadzymes). In many embodiments, a targeting moiety will comprise apolypeptide or polypeptide complex, and particularly an antibody orpolypeptide(s) including an antigen binding site of an antibody. Forexample, a targeting moiety may include a monoclonal antibody, an Faband F(ab)2, an scFv, a heavy chain variable region and a light chainvariable region. Optionally, the targeting moiety is an artificialprotein or peptide sequence engineered to bind to the substrate. Incertain embodiments, the targeting moiety is a polyanionic orpolycationic binding agent. Optionally, the targeting moiety is anoligonucleotide, a polysaccharide or a lectin. In certain embodiments,the substrate is a receptor, and the targeting moiety includes a ligand(or binding portion thereof) that binds to the receptor. In certainembodiments, the substrate is a ligand of a receptor, and the targetingmoiety includes a ligand binding portion of the receptor, particularly asoluble ligand binding portion.

[0015] An adzyme may be used to target essentially any amenablesubstrate in a variety of technological applications, includingtherapeutic uses, industrial uses, environmental uses and uses inmicrofabrications. In a preferred embodiment, an adzyme substrate isfrom a mammal, such as a rodent, a non-human primate or a human. In apreferred embodiment, the substrate is endogenous to a human patient. Incertain embodiments, the substrate is a biomolecule produced by a cell,such as a polypeptide, a polysaccharide, a nucleic acid, a lipid, or asmall molecule. In certain embodiments, the substrate is a diffusibleextracellular molecule, and preferably an extracellular signalingmolecule that may act on an extracellular or intracellular receptor totriggers receptor-mediated cellular signaling. Optionally, theextracellular signaling molecule is an extracellular polypeptidesignaling molecule, such as an inflammatory cytokine. In a preferredembodiment, the substrate is an interleukin-1 (e.g., IL-1α, IL-1β) orTNF-α. In certain embodiments, the substrate is a polypeptide hormone, agrowth factor and/or a cytokine, especially an inflammatory cytokine.Optionally, the adzyme acts to reduce a pro-inflammatory activity of asubstrate. A substrate may be selected from among the following:four-helix bundle factors, EGF-like factors, insulin-like factors,P-trefoil factors and cysteine knot factors. In certain embodiments, thesubstrate is a receptor, particularly a receptor with some portionexposed to the extracellular surface. Optionally, the substrate is aunique receptor subunit of a heteromeric receptor complex. In certainembodiments, the substrate is a biomolecules that is a component of abiomolecular accretion, such as an amyloid deposit or an atheroscleroticplaque. In certain embodiments, the substrate is an intracellularbiomolecule, and in such instances, it may be desirable to use an adzymethat is able to enter the targeted cells, such as an adzyme that furthercomprises a transcytosis moiety that promotes transcytosis of the adzymeinto the cell. In certain embodiments, the substrate is a biomoleculeproduced by a pathogen, such as a protozoan, a fungus, a bacterium or avirus. The substrate may be a prion protein. In a preferred embodiment,the substrate is endogenous to a human patient. In such an embodiment,the adzyme is preferably effective against the substrate in the presenceof physiological levels of an abundant human serum protein, such as,serum albumins or an abundant globin.

[0016] In a preferred embodiment, the substrate for an adzyme is TNFα.In the case of a direct adzyme, the targeting moiety binds to TNFα.Preferably, the catalytic domain comprises a protease that decreasesTNFα activity. For example, the protease is may be selected from among:MT1-MMP; MMP12; tryptase; MT2-MMP; elastase; MMP7; chymotrypsin; andtrypsin. The targeting moiety may be selected from among, a solubleportion of a TNFα receptor and a single chain antibody that binds toTNFα, although other targeting moieties are possible. A preferredtargeting moiety is an sp55 portion of TNFα Receptor 1 (TNFR1).

[0017] In another preferred embodiment, the substrate for an adzyme isan interleukin-1, such as IL-1α or IL-1β. In the case of a directadzyme, the targeting moiety binds to the interleukin-1 substrate.Preferably, the catalytic domain comprises a protease that decreases anIL-1 bioactivity.

[0018] In one aspect, the invention provides an adzyme for enzymaticallyaltering a substrate, the adzyme comprising: a catalytic domain thatcatalyzes a chemical reaction converting said substrate to one or moreproducts, and a targeting moiety that reversibly binds with an addresssite on said substrate or with an address site on a second molecule thatoccurs in functional proximity to the substrate, wherein said targetingmoiety and said catalytic domain are heterologous with respect to eachother, said targeting moiety, when provided separately, binds to thesubstrate, said catalytic domain, when provided separately, catalyzesthe chemical reaction converting said substrate to one or more products,and said adzyme has one or more desirable properties, with respect tothe reaction with said substrate. For example, in this aspect, theadzyme may have a potency at least 2 times greater than the catalyticdomain or the targeting moiety alone, and preferably at least 3, 5, 10,20 or more times greater than the potency of the catalytic domain ortargeting moiety alone. The adzyme may have a k_(on) of 10³ M⁻¹s⁻¹ orgreater, and optionally a k_(on) of 10⁴ M⁻¹s⁻¹, 10⁵ M⁻¹s⁻¹, 10⁶ M⁻¹s⁻¹,10⁷ M⁻¹s⁻¹ or greater. The adzyme may have a k_(cat) of 0.1 sec⁻¹ orgreater, and optionally a k_(cat) of 1 sec⁻¹, 10 sec⁻¹, 50 sec⁻¹ orgreater. The adzyme may have a K_(D) that is at least 5, 10, 25, 50 or100 or more fold less than the K_(m) of the catalytic domain. The adzymemay have a k_(off) of 10⁻⁴ sec⁻¹ or greater, and optionally a k_(off) of10⁻³ sec⁻¹, k_(off) of 10⁻² sec⁻¹, or greater. The adzyme may have acatalytic efficiency that is at least 5 fold greater than the catalyticefficiency of the catalytic domain alone, and optionally a catalyticefficiency that is at least 10 fold, 20 fold, 50 fold or 100 foldgreater than that of the catalytic domain. The adzyme may have a K_(m)at least 5 fold, 10 fold, 20 fold, 50 fold, or 100 fold less than theK_(m) of the catalytic domain alone. The adzyme may have an effectivesubstrate concentration that is at least 5 fold, 10 fold, 20 fold, 50fold or 100 fold greater than the actual substrate concentration. Incertain preferred embodiments, the adzyme will be designed so as tocombine two or more of the above described properties.

[0019] In certain embodiments of an adzyme having one or more of suchproperties with respect to the reaction with the substrate molecule, acatalytic domain and a targeting domain of the adzyme are joined by apolypeptide and linker to form a fusion protein. The fusion protein maybe generated in a variety of ways, including chemical coupling andcotranslation. In a preferred embodiment, the fusion protein is acotranslational fusion protein encoded by a recombinant nucleic acid. Incertain embodiments the linker for the fusion protein is an unstructuredpeptide. Optionally, the linker includes one or more repeats of Ser₄Glyor SerGly₄. In preferred embodiments, the linker is selected to providesteric geometry between said catalytic domain and said targeting moietysuch that said adzyme is more effective against the substrate thaneither the catalytic domain or targeting moiety alone. For example, thelinker may be selected such that the adzyme is more potent than saidcatalytic domain or targeting moiety with respect to the reaction withsaid substrate. The linker may be selected such that the targetingmoiety presents the substrate to the enzymatic domain at an effectiveconcentration at least 5 fold greater than would be present in theabsence of the targeting moiety.

[0020] In certain embodiments of an adzyme having one or more of suchproperties, the adzyme is an immunoglobulin fusion, wherein thecatalytic domain and the targeting moiety are joined, in a geometryconsistent with effectiveness against substrate, to at least a portionof an immunoglobulin comprising a constant domain of an immunoglobulin.For example, the adzyme may comprise a first fusion protein and a secondfusion protein, wherein the first fusion protein comprises a constantportion of an immunoglobulin heavy chain and a catalytic domain, andwherein the second fusion protein comprises a constant portion of animmunoglobulin heavy chain and a targeting domain that reversibly bindswith an address site on or in functional proximity to the substrate.Preferably the immunoglobulin portions are Fc portions that dimerize bydisulfide bonds.

[0021] In certain embodiments of an adzyme having one or more of suchproperties, a catalytic domain may include essentially any enzymaticdomain that achieves the desired effect on a selected substrate. Thecatalytic domain may be selected so as to modify one or more pendantgroups of said substrate. The substrate may include a chiral atom, andsaid catalytic domain may alter the ratio of stereoisomers. Thecatalytic domain may alter the level of post-translational modificationof the polypeptide substrate, such as a glycosylation, phosphorylation,sulfation, fatty acid modification, alkylation, prenylation oracylation. Examples of enzymatic domains that may be selected include: aprotease, an esterase, an amidase, a lactamase, a cellulase, an oxidase,an oxidoreductase, a reductase, a transferase, a hydrolase, anisomerase, a ligase, a lipase, a phospholipase, a phosphatase, a kinase,a sulfatase, a lysozyme, a glycosidase, a nuclease, an aldolase, aketolase, a lyase, a cyclase, a reverse transcriptase, a hyaluronidase,an amylase, a cerebrosidase and a chitinase. Regardless of the type ofcatalytic domain, it may be desirable that the adzyme be resistant toautocatalysis (e.g., inter- or intra-molecular reactions), particularlyat an adzyme concentration that is about equal to the concentration ofadzyme in a solution to be administered to a subject. In certainembodiment, the adzyme acts on the substrate such that a product of thechemical reaction is an antagonist of the substrate.

[0022] In certain preferred embodiments of an adzyme having one or moreof the properties described above with respect to the reaction with thesubstrate, the adzyme includes a protease domain that, when active,cleaves at least one peptide bond of a polypeptide substrate. In generalit will be desirable to design the adzyme such that it is resistant tocleavage by the protease catalytic domain. The protease domain may begenerated as a zymogen (an inactive form) and then activated prior touse. The adzyme may be purified from a cell culture in the presence of areversible protease inhibitor, and such inhibitor may be included in anysubsequent processing or storage activities.

[0023] In certain embodiments of an adzyme having one or more of theproperties described above with respect to the reaction with thesubstrate, a targeting moiety may include essentially any molecule orassembly of molecules that binds to the address site (e.g., on thesubstrate in the case of direct adzymes or on a molecule that occurs infunctional proximity to the substrate, in the case of proximityadzymes). In many embodiments, a targeting moiety will comprise apolypeptide or polypeptide complex, and particularly an antibody orpolypeptide(s) including an antigen binding site of an antibody. Forexample, a targeting moiety may include a monoclonal antibody, an Faband F(ab)2, an scFv, a heavy chain variable region and a light chainvariable region. Optionally, the targeting moiety is an artificialprotein or peptide sequence engineered to bind to the substrate. Incertain embodiments, the targeting moiety is a polyanionic orpolycationic binding agent. Optionally, the targeting moiety is anoligonucleotide, a polysaccharide or a lectin. In certain embodiments,the substrate is a receptor, and the targeting moiety includes a ligand(or binding portion thereof) that binds to the receptor. In certainembodiments, the substrate is a ligand of a receptor, and the targetingmoiety includes a ligand binding portion of the receptor, particularly asoluble ligand binding portion.

[0024] In certain embodiments of an adzyme having one or more of theproperties described above with respect to the reaction with thesubstrate, the substrate is a biomolecule produced by a cell, such as apolypeptide, a polysaccharide, a nucleic acid, a lipid, or a smallmolecule. In certain embodiments, the substrate is a diffusibleextracellular molecule, and preferably an extracellular signalingmolecule that may act on an extracellular or intracellular receptor totriggers receptor-mediated cellular signaling. Optionally, theextracellular signaling molecule is an extracellular polypeptidesignaling molecule, such as an inflammatory cytokine. In a preferredembodiment, the substrate is an interleukin-1 (e.g., IL-1α, IL-1β) or aTNF-α. In certain embodiments, the substrate is a polypeptide hormone, agrowth factor and/or a cytokine, especially an inflammatory cytokine.Optionally, the adzyme acts to reduces a pro-inflammatory activity of asubstrate. A substrate may be selected from is selected from the groupconsisting of four-helix bundle factors, EGF-like factors, insulin-likefactors, β-trefoil factors and cysteine knot factors. In certainembodiments, the substrate is a receptor, particularly a receptor withsome portion exposed to the extracellular surface. Optionally, thesubstrate is a unique receptor subunit of a heteromeric receptorcomplex. In certain embodiments, the biomolecule is a component of abiomolecular accretion, such as an amyloid deposit or an atheroscleroticplaque. In certain embodiments, the substrate is an intracellularbiomolecule, and in such instances, it may be desirable to use an adzymethat is able to enter the targeted cells, such as an adzyme that furthercomprises a transcytosis moiety that promotes transcytosis of the adzymeinto the cell. In certain embodiments, the substrate is a biomoleculeproduced by a pathogen, such as a protozoan, a fungus, a bacterium or avirus. The substrate may be a prion protein. In a preferred embodiment,the substrate is endogenous to a human patient. In such an embodiment,the adzyme is preferably effective against the substrate in the presenceof physiological levels of an abundant human serum protein, such as,serum albumins or an abundant globin.

[0025] In a preferred embodiment of an adzyme having one or more of theproperties described above with respect to the reaction with thesubstrate, the substrate for an adzyme is TNFα. In the case of a directadzyme, the targeting moiety binds to TNFα. Preferably, the catalyticdomain comprises a protease that decreases TNFα activity. For example,the protease is may be selected from among: MT1-MMP; MMP12; tryptase;MT2-MMP; elastase; MMP7; chymotrypsin; and trypsin. The targeting moietymay be selected from among, a soluble portion of a TNFα receptor and asingle chain antibody that binds to TNFα, although other targetingmoieties are possible. A preferred targeting moiety is an sp55 portionof TNFα Receptor 1 (TNFR1).

[0026] In another preferred embodiment of an adzyme having one or moreof the properties described above with respect to the reaction with thesubstrate, the substrate for an adzyme is an interleukin-1, such asIL-1α or IL-1β. In the case of a direct adzyme, the targeting moietybinds to the interleukin-1 substrate. Preferably, the catalytic domaincomprises a protease that decreases an IL-1 bioactivity.

[0027] In one aspect, the invention provides an adzyme for enzymaticallyaltering a substrate, the adzyme comprising: a catalytic domain thatcatalyzes a chemical reaction converting said substrate to one or moreproducts, and a targeting moiety that reversibly binds with an addresssite on said substrate or with an address site on a second molecule thatoccurs in functional proximity to the substrate, wherein the substrateis an extracellular signaling molecule, said targeting moiety and saidcatalytic domain are heterologous with respect to each other, saidtargeting moiety, when provided separately, binds to the substrate, saidcatalytic domain, when provided separately, catalyzes the chemicalreaction converting said substrate to one or more products, and saidchimeric protein construct is more potent than said catalytic domain ortargeting moiety with respect to the reaction with said substrate.

[0028] In certain embodiments of an adzyme that targets an extracellularsignaling molecule, a catalytic domain and a targeting domain of theadzyme are joined by a polypeptide and linker to form a fusion protein.A fusion protein may be generated in a variety of ways, includingchemical coupling and cotranslation. In a preferred embodiment, thefusion protein is a cotranslational fusion protein encoded by arecombinant nucleic acid. In certain embodiments the linker for thefusion protein is an unstructured peptide. Optionally, the linkerincludes one or more repeats of Ser₄Gly or SerGly₄. In preferredembodiments, the linker is selected to provide steric geometry betweensaid catalytic domain and said targeting moiety such that said adzyme ismore effective against the substrate than either the catalytic domain ortargeting moiety alone. For example, the linker may be selected suchthat the adzyme is more potent than said catalytic domain or targetingmoiety with respect to the reaction with said substrate. The linker maybe selected such that the targeting moiety presents the substrate to theenzymatic domain at an effective concentration at least 5 fold greaterthan would be present in the absence of the targeting moiety.

[0029] In certain embodiments of an adzyme that targets an extracellularsignaling molecule, the adzyme is an immunoglobulin fusion, wherein thecatalytic domain and the targeting moiety are joined, in a geometryconsistent with effectiveness against substrate, to at least a portionof an immunoglobulin comprising a constant domain of an immunoglobulin.For example, the adzyme may comprise a first fusion protein and a secondfusion protein, wherein the first fusion protein comprises a constantportion of an immunoglobulin heavy chain and a catalytic domain, andwherein the second fusion protein comprises a constant portion of animmunoglobulin heavy chain and a targeting domain that reversibly bindswith an address site on or in functional proximity to the substrate.Preferably the immunoglobulin portions are Fc portions that dimerize bydisulfide bonds.

[0030] In certain embodiments of an adzyme that targets an extracellularsignaling molecule, the adzyme is designed so as to have one or moredesirable properties with respect to the reaction with said substrate.In many instances, such properties will be significant for achieving thedesired effect of the adzyme on the substrate. For example, an adzymemay have a potency at least 2 times greater than the catalytic domain orthe targeting moiety alone, and preferably at least 3, 5, 10, 20 or moretimes greater than the potency of the catalytic domain or targetingmoiety alone. The adzyme may have a k_(on) of 10³ M⁻¹s⁻¹ or greater, andoptionally a k_(on) of 10⁴ M⁻¹s⁻, 10⁵ M⁻¹s⁻¹, 10⁶ M⁻¹s⁻¹ _(, 10) ⁷M⁻¹s⁻¹ or greater. The adzyme may have a k_(cat) of 0.1 sec⁻¹ orgreater, and optionally a k_(cat) of 1 sec⁻¹, 10 sec⁻¹, 50 sec⁻¹ orgreater. The adzyme may have a K_(D) that is at least 5, 10, 25, 50 or100 or more fold less than the K_(m) of the catalytic domain. The adzymemay have a k_(off) of 10⁻⁴ sec⁻¹ or greater, and optionally a k_(off) of10⁻² sec⁻¹, k_(off) of 10⁻² sec⁻¹, or greater. The adzyme may have acatalytic efficiency that is at least 5 fold greater than the catalyticefficiency of the catalytic domain alone, and optionally a catalyticefficiency that is at least 10 fold, 20 fold, 50 fold or 100 foldgreater than that of the catalytic domain. The adzyme may have a K_(m)at least 5 fold, 10 fold, 20 fold, 50 fold, or 100 fold less than theK_(m) of the catalytic domain alone. The adzyme may have an effectivesubstrate concentration that is at least 5 fold, 10 fold, 20 fold, 50fold or 100 fold greater than the actual substrate concentration. Incertain preferred embodiments, an adzyme will be designed so as tocombine two or more of the above described properties.

[0031] In certain embodiments of an adzyme that targets an extracellularsignaling molecule, a catalytic domain may include essentially anyenzymatic domain that achieves the desired effect on a selectedsubstrate. The catalytic domain may be selected so as to modify one ormore pendant groups of said substrate. The substrate may include achiral atom, and said catalytic domain may alter the ratio ofstereoisomers. The catalytic domain may alter the level ofpost-translational modification of a polypeptide substrate, such as aglycosylation, phosphorylation, sulfation, fatty acid modification,alkylation, prenylation or acylation. Examples of enzymatic domains thatmay be selected include: a protease, an esterase, an amidase, alactamase, a cellulase, an oxidase, an oxidoreductase, a reductase, atransferase, a hydrolase, an isomerase, a ligase, a lipase, aphospholipase, a phosphatase, a kinase, a sulfatase, a lysozyme, aglycosidase, a nuclease, an aldolase, a ketolase, a lyase, a cyclase, areverse transcriptase, a hyaluronidase, an amylase, a cerebrosidase anda chitinase. Regardless of the type of catalytic domain, it may bedesirable that the adzyme be resistant to autocatalysis (e.g., inter- orintra-molecular reactions), particularly at an adzyme concentration thatis about equal to the concentration of adzyme in a solution to beadministered to a subject. In certain embodiment, the adzyme acts on thesubstrate such that a product of the chemical reaction is an antagonistof the substrate. In certain embodiment, the adzyme acts on thesubstrate such that a product of the chemical reaction is an antagonistof the substrate.

[0032] In certain preferred embodiments of an adzyme that targets anextracellular signaling molecule, the adzyme includes a protease domainthat, when active, cleaves at least one peptide bond of a polypeptidesubstrate. In general it will be desirable to design the adzyme suchthat it is resistant to cleavage by the protease catalytic domain. Theprotease domain may be generated as a zymogen (an inactive form) andthen activated prior to use. The adzyme may be purified from a cellculture in the presence of a reversible protease inhibitor, and suchinhibitor may be included in any subsequent processing or storageactivities.

[0033] In certain embodiments of an adzyme that targets an extracellularsignaling molecule, a targeting moiety may include essentially anymolecule or assembly of molecules that binds to the address site (e.g.,on the substrate in the case of direct adzymes or on a molecule thatoccurs in functional proximity to the substrate, in the case ofproximity adzymes). In many embodiments, a targeting moiety willcomprise a polypeptide or polypeptide complex, and particularly anantibody or polypeptide(s) including an antigen binding site of anantibody. For example, a targeting moiety may include a monoclonalantibody, an Fab and F(ab)2, an scFv, a heavy chain variable region anda light chain variable region. Optionally, the targeting moiety is anartificial protein or peptide sequence engineered to bind to thesubstrate. In certain embodiments, the targeting moiety is a polyanionicor polycationic binding agent. Optionally, the targeting moiety is anoligonucleotide, a polysaccharide or a lectin. In certain embodiments,the substrate is a ligand of a receptor, and the targeting moietyincludes a ligand binding portion of the receptor, particularly asoluble ligand binding portion.

[0034] In certain embodiments of an adzyme targeted to an extracellularsignaling molecule, the substrate is preferably an extracellularsignaling molecule that acts on an extracellular or intracellularreceptor to triggers receptor-mediated cellular signaling. Optionally,the extracellular signaling molecule is an extracellular polypeptidesignaling molecule, such as an inflammatory cytokine. In a preferredembodiment, the substrate is an interleukin-1 (e.g., IL-1α, IL-1β) or aTNF-α. In certain embodiments, the substrate is a polypeptide hormone, agrowth factor and/or a cytokine, especially an inflammatory cytokine.Optionally, the adzyme acts to reduces a pro-inflammatory activity of asubstrate. A substrate may be selected from is selected from the groupconsisting of four-helix bundle factors, EGF-like factors, insulin-likefactors, β-trefoil factors and cysteine knot factors. In a preferredembodiment, the substrate is endogenous to a human patient. In such anembodiment, the adzyme is preferably effective against the substrate inthe presence of physiological levels of an abundant human serum protein,such as, serum albumins or an abundant globin.

[0035] In a preferred embodiment of an adzyme targeted to anextracellular signaling molecule, the substrate for the adzyme is TNFα.In the case of a direct adzyme, the targeting moiety binds to TNFα.Preferably, the catalytic domain comprises a protease that decreasesTNFα activity. For example, the protease is may be selected from among:MT1-MMP; MMP12; tryptase; MT2-MMP; elastase; MMP7; chymotrypsin; andtrypsin. The targeting moiety may be selected from among, a solubleportion of a TNFα receptor and a single chain antibody that binds toTNFα, although other targeting moieties are possible. A preferredtargeting moiety is an sp55 portion of TNFα Receptor 1 (TNFR1).

[0036] In another preferred embodiment of an adzyme targeted to anextracellular signaling molecule, the substrate for an adzyme is aninterleukin-1, such as IL-1α or IL-1β. In the case of a direct adzyme,the targeting moiety binds to the interleukin-1 substrate. Preferably,the catalytic domain comprises a protease that decreases an IL-1bioactivity.

[0037] In one aspect, the invention provides adzymes for enzymaticallyaltering a substrate, the adzyme comprising a polypeptide comprising: acatalytic domain that catalyzes a chemical reaction converting saidsubstrate to one or more products, a targeting domain that reversiblybinds with an address site on said substrate or with an address site ona second molecule that occurs in functional proximity to the substrate,and a linker joining said catalytic domain and said targeting domain,wherein said substrate is a receptor, said targeting moiety and saidcatalytic domain are heterologous with respect to each other, saidtargeting domain, when provided separately, binds to the substrate, saidcatalytic domain, when provided separately, catalyzes the chemicalreaction converting said substrate to one or more products, and saidchimeric protein construct is more potent than said catalytic domain ortargeting moiety with respect to the reaction with said substrate.

[0038] In certain embodiments of an adzyme that targets a receptor, acatalytic domain and a targeting domain of the adzyme are joined by apolypeptide and linker to form a fusion protein. A fusion protein may begenerated in a variety of ways, including chemical coupling andcotranslation. In a preferred embodiment, the fusion protein is acotranslational fusion protein encoded by a recombinant nucleic acid. Incertain embodiments the linker for the fusion protein is an unstructuredpeptide. Optionally, the linker includes one or more repeats of Ser₄Glyor SerGly₄. In preferred embodiments, the linker is selected to providesteric geometry between said catalytic domain and said targeting moietysuch that said adzyme is more effective against the substrate thaneither the catalytic domain or targeting moiety alone. For example, thelinker may be selected such that the adzyme is more potent than saidcatalytic domain or targeting moiety with respect to the reaction withsaid substrate. The linker may be selected such that the targetingmoiety presents the substrate to the enzymatic domain at an effectiveconcentration at least 5 fold greater than would be present in theabsence of the targeting moiety.

[0039] In certain embodiments of an adzyme that targets a receptor, theadzyme is an immunoglobulin fusion, wherein the catalytic domain and thetargeting moiety are joined, in a geometry consistent with effectivenessagainst substrate, to at least a portion of an immunoglobulin comprisinga constant domain of an immunoglobulin. For example, the adzyme maycomprise a first fusion protein and a second fusion protein, wherein thefirst fusion protein comprises a constant portion of an immunoglobulinheavy chain and a catalytic domain, and wherein the second fusionprotein comprises a constant portion of an immunoglobulin heavy chainand a targeting domain that reversibly binds with an address site on orin functional proximity to the substrate. Preferably the immunoglobulinportions are Fc portions that dimerize by disulfide bonds.

[0040] In certain embodiments of an adzyme that targets a receptor, theadzyme is designed so as to have one or more desirable properties adzymehas one or more desirable properties, with respect to the reaction withsaid substrate. In many instances, such properties will be significantfor achieving the desired effect of the adzyme on the substrate. Forexample, an adzyme may have a potency at least 2 times greater than thecatalytic domain or the targeting moiety alone, and preferably at least3, 5, 10, 20 or more times greater than the potency of the catalyticdomain or targeting moiety alone. The adzyme may have a k_(on) of 10³M⁻¹s⁻¹ or greater, and optionally a k_(on) of 10⁴ M⁻¹s⁻, 10⁵ M⁻¹s⁻¹, 10⁶M⁻¹s⁻, 10⁷ M⁻¹s⁻¹ or greater. The adzyme may have a k_(cat) of 0.1 sec⁻¹or greater, and optionally a k_(cat) of 1 sec⁻¹, 10 sec⁻¹, 50 sec⁻¹ orgreater. The adzyme may have a K_(D) that is at least 5, 10, 25, 50 or100 or more fold less than the K_(m) of the catalytic domain. The adzymemay have a k_(off) of 10⁴ sec⁻¹ or greater, and optionally a k_(off) of10⁻² sec¹, k_(off) of 10⁻² sec⁻¹, or greater. The adzyme may have acatalytic efficiency that is at least 5 fold greater than the catalyticefficiency of the catalytic domain alone, and optionally a catalyticefficiency that is at least 10 fold, 20 fold, 50 fold or 100 foldgreater than that of the catalytic domain. The adzyme may have a K_(m)at least 5 fold, 10 fold, 20 fold, 50 fold, or 100 fold less than theK_(m) of the catalytic domain alone. The adzyme may have an effectivesubstrate concentration that is at least 5 fold, 10 fold, 20 fold, 50fold or 100 fold greater than the actual substrate concentration. Incertain preferred embodiments, an adzyme will be designed so as tocombine two or more of the above described properties.

[0041] In certain embodiments of an adzyme that targets a receptor, acatalytic domain may include essentially any enzymatic domain thatachieves the desired effect on a selected substrate. The catalyticdomain may be selected so as to modify one or more pendant groups ofsaid substrate. The substrate may include a chiral atom, and saidcatalytic domain may alter the ratio of stereoisomers. The catalyticdomain may alter the level of post-translational modification of apolypeptide substrate, such as a glycosylation, phosphorylation,sulfation, fatty acid modification, alkylation, prenylation oracylation. Examples of enzymatic domains that may be selected include: aprotease, an esterase, an amidase, a lactamase, a cellulase, an oxidase,an oxidoreductase, a reductase, a transferase, a hydrolase, anisomerase, a ligase, a lipase, a phospholipase, a phosphatase, a kinase,a sulfatase, a lysozyme, a glycosidase, a nuclease, an aldolase, aketolase, a lyase, a cyclase, a reverse transcriptase, a hyaluronidase,an amylase, a cerebrosidase and a chitinase. Regardless of the type ofcatalytic domain, it may be desirable that the adzyme be resistant toautocatalysis (e.g., inter- or intra-molecular reactions), particularlyat an adzyme concentration that is about equal to the concentration ofadzyme in a solution to be administered to a subject. In certainembodiment, the adzyme acts on the substrate such that a product of thechemical reaction is an antagonist of the substrate. In certainembodiment, the adzyme acts on the substrate such that a product of thechemical reaction is an antagonist of the substrate.

[0042] In certain preferred embodiments of an adzyme that targets areceptor, the adzyme includes a protease domain that, when active,cleaves at least one peptide bond of a polypeptide substrate. In generalit will be desirable to design the adzyme such that it is resistant tocleavage by the protease catalytic domain. The protease domain may begenerated as a zymogen (an inactive form) and then activated prior touse. The adzyme may be purified from a cell culture in the presence of areversible protease inhibitor, and such inhibitor may be included in anysubsequent processing or storage activities.

[0043] In certain embodiments of an adzyme that targets a receptor, atargeting moiety may include essentially any molecule or assembly ofmolecules that binds to the address site (e.g., on the substrate in thecase of direct adzymes or on a molecule that occurs in functionalproximity to the substrate, in the case of proximity adzymes). In manyembodiments, a targeting moiety will comprise a polypeptide orpolypeptide complex, and particularly an antibody or polypeptide(s)including an antigen binding site of an antibody. For example, atargeting moiety may include a monoclonal antibody, an Fab and F(ab)2,an scFv, a heavy chain variable region and a light chain variableregion. Optionally, the targeting moiety is an artificial protein orpeptide sequence engineered to bind to the substrate. In certainembodiments, the targeting moiety is a polyanionic or polycationicbinding agent. Optionally, the targeting moiety is an oligonucleotide, apolysaccharide or a lectin. The targeting moiety may include a ligand(or binding portion thereof) that binds to the receptor.

[0044] In certain embodiments of an adzyme that targets a receptor, thesubstrate is a receptor with some portion exposed to the extracellularsurface. Optionally, the substrate is a unique receptor subunit of aheteromeric receptor complex. In a preferred embodiment, the substrateis endogenous to a human patient. In such an embodiment, the adzyme ispreferably effective against the substrate in the presence ofphysiological levels of an abundant human serum protein, such as, serumalbumins or an abundant globin.

[0045] In a preferred embodiment of an adzyme targeted to a receptor,the substrate for the adzyme is a TNFα receptor, such as TNFR1 or TNFR2.In the case of a direct adzyme, the targeting moiety binds to thereceptor. Preferably, the catalytic domain comprises a protease thatdecreases the TNFα stimulated activity of thereceptor. The targetingmoiety may be selected from among, a receptor binding portion of TNFαand a single chain antibody that binds to the receptor, although othertargeting moieties are possible.

[0046] In another preferred embodiment of an adzyme targeted to areceptor, the substrate for an adzyme is an interleukin-1 receptor(IL-1R). In the case of a direct adzyme, the targeting moiety binds tothe IL-1R. Preferably, the catalytic domain comprises a protease thatdecreases an IL-1R bioactivity.

[0047] In a further aspect, the invention provides an adzyme forenzymatically altering a substrate, the adzyme comprising: a catalyticdomain that catalyzes a chemical reaction converting said substrate toone or more products, and a targeting moiety that reversibly binds withan address site on said substrate or with an address site on a secondmolecule that occurs in functional proximity to the substrate, whereinone or more of said products is an antagonist of an activity of saidsubstrate.

[0048] In certain embodiments of an adzyme that generates an antagonistof the substrate, a catalytic domain and a targeting domain of theadzyme are joined by a polypeptide and linker to form a fusion protein.A fusion protein may be generated in a variety of ways, includingchemical coupling and cotranslation. In a preferred embodiment, thefusion protein is a cotranslational fusion protein encoded by arecombinant nucleic acid. In certain embodiments the linker for thefusion protein is an unstructured peptide. Optionally, the linkerincludes one or more repeats of Ser₄Gly or SerGly₄. In preferredembodiments, the linker is selected to provide steric geometry betweensaid catalytic domain and said targeting moiety such that said adzyme ismore effective against the substrate than either the catalytic domain ortargeting moiety alone. For example, the linker may be selected suchthat the adzyme is more potent than said catalytic domain or targetingmoiety with respect to the reaction with said substrate. The linker maybe selected such that the targeting moiety presents the substrate to theenzymatic domain at an effective concentration at least 5 fold greaterthan would be present in the absence of the targeting moiety.

[0049] In certain embodiments of an adzyme that generates an antagonistof the substrate, the adzyme is an immunoglobulin fusion, wherein thecatalytic domain and the targeting moiety are joined, in a geometryconsistent with effectiveness against substrate, to at least a portionof an immunoglobulin comprising a constant domain of an immunoglobulin.For example, the adzyme may comprise a first fusion protein and a secondfusion protein, wherein the first fusion protein comprises a constantportion of an immunoglobulin heavy chain and a catalytic domain, andwherein the second fusion protein comprises a constant portion of animmunoglobulin heavy chain and a targeting domain that reversibly bindswith an address site on or in functional proximity to the substrate.Preferably the immunoglobulin portions are Fc portions that dimerize bydisulfide bonds.

[0050] In certain embodiments of an adzyme that generates an antagonistof the substrate, the adzyme is designed so as to have one or moredesirable properties adzyme has one or more desirable properties, withrespect to the reaction with said substrate. In many instances, suchproperties will be significant for achieving the desired effect of theadzyme on the substrate. For example, an adzyme may have a potency atleast 2 times greater than the catalytic domain or the targeting moietyalone, and preferably at least 3, 5, 10, 20 or more times greater thanthe potency of the catalytic domain or targeting moiety alone. Theadzyme may have a k_(on) of 10³ M⁻¹s⁻¹ or greater, and optionally ak_(on) of 10⁴ M⁻¹s⁻¹, 10⁵ M⁻¹s⁻, 10⁶ M⁻¹s⁻¹, 10⁷ M⁻s⁻¹ or greater. Theadzyme may have a k_(cat) of 0.1 sec⁻¹ or greater, and optionally ak_(cat) of 1 sec⁻¹, 10 sec⁻¹, 50 sec⁻¹ or greater. The adzyme may have aK_(D) that is at least 5, 10, 25, 50 or 100 or more fold less than theK_(m) of the catalytic domain. The adzyme may have a k_(off) of 10⁻⁴sec⁻¹ or greater, and optionally a k_(off) of 10⁻² sec⁻¹, k_(off) of10⁻² sec⁻¹, or greater. The adzyme may have a catalytic efficiency thatis at least 5 fold greater than the catalytic efficiency of thecatalytic domain alone, and optionally a catalytic efficiency that is atleast 10 fold, 20 fold, 50 fold or 100 fold greater than that of thecatalytic domain. The adzyme may have a K_(m) at least 5 fold, 10 fold,20 fold, 50 fold, or 100 fold less than the K_(m) of the catalyticdomain alone. The adzyme may have an effective substrate concentrationthat is at least 5 fold, 10 fold, 20 fold, 50 fold or 100 fold greaterthan the actual substrate concentration. In certain preferredembodiments, an adzyme will be designed so as to combine two or more ofthe above described properties.

[0051] In certain embodiments of an adzyme that generates an antagonistof the substrate, a catalytic domain may include essentially anyenzymatic domain that achieves the desired effect on a selectedsubstrate. The catalytic domain may be selected so as to modify one ormore pendant groups of said substrate. The substrate may include achiral atom, and said catalytic domain may alter the ratio ofstereoisomers. The catalytic domain may alter the level ofpost-translational modification of a polypeptide substrate, such as aglycosylation, phosphorylation, sulfation, fatty acid modification,alkylation, prenylation or acylation. Examples of enzymatic domains thatmay be selected include: a protease, an esterase, an amidase, alactamase, a cellulase, an oxidase, an oxidoreductase, a reductase, atransferase, a hydrolase, an isomerase, a ligase, a lipase, aphospholipase, a phosphatase, a kinase, a sulfatase, a lysozyme, aglycosidase, a nuclease, an aldolase, a ketolase, a lyase, a cyclase, areverse transcriptase, a hyaluronidase, an amylase, a cerebrosidase anda chitinase. Regardless of the type of catalytic domain, it may bedesirable that the adzyme be resistant to autocatalysis (e.g., inter- orintra-molecular reactions), particularly at an adzyme concentration thatis about equal to the concentration of adzyme in a solution to beadministered to a subject. In certain embodiment, the adzyme acts on thesubstrate such that a product of the chemical reaction is an antagonistof the substrate. In certain embodiment, the adzyme acts on thesubstrate such that a product of the chemical reaction is an antagonistof the substrate.

[0052] In certain preferred embodiments of an adzyme that generates anantagonist of the substrate, the adzyme includes a protease domain that,when active, cleaves at least one peptide bond of a polypeptidesubstrate. In general it will be desirable to design the adzyme suchthat it is resistant to cleavage by the protease catalytic domain. Theprotease domain may be generated as a zymogen (an inactive form) andthen activated prior to use. The adzyme may be purified from a cellculture in the presence of a reversible protease inhibitor, and suchinhibitor may be included in any subsequent processing or storageactivities.

[0053] In certain embodiments of an adzyme that generates an antagonistof the substrate, a targeting moiety may include essentially any molecule or assembly of molecules that binds to the address site (e.g.,on the substrate in the case of direct adzymes or on a molecule thatoccurs in functional proximity to the substrate, in the case ofproximity adzymes). In many embodiments, a targeting moiety willcomprise a polypeptide or polypeptide complex, and particularly anantibody or polypeptide(s) including an antigen binding site of anantibody. For example, a targeting moiety may include a monoclonalantibody, an Fab and F(ab)2, an scFv, a heavy chain variable region anda light chain variable region. Optionally, the targeting moiety is anartificial protein or peptide sequence engineered to bind to thesubstrate. In certain embodiments, the targeting moiety is a polyanionicor polycationic binding agent. Optionally, the targeting moiety is anoligonucleotide, a polysaccharide or a lectin. The targeting moiety mayinclude a ligand (or binding portion thereof) that binds to thereceptor.

[0054] In certain embodiments of an adzyme that generates an antagonistof the substrate, the substrate is a biomolecule produced by a cell,such as a polypeptide, a polysaccharide, a nucleic acid, a lipid, or asmall molecule. In certain embodiments, the substrate is a diffusibleextracellular molecule, and preferably an extracellular signalingmolecule that may act on an extracellular or intracellular receptor totriggers receptor-mediated cellular signaling. Optionally, theextracellular signaling molecule is an extracellular polypeptidesignaling molecule, such as an inflammatory cytokine. In a preferredembodiment, the substrate is an interleukin-1 (e.g., IL-1α, IL-1β) or aTNF-α. In certain embodiments, the substrate is a polypeptide hormone, agrowth factor and/or a cytokine, especially an inflammatory cytokine.Optionally, the adzyme acts to reduces a pro-inflammatory activity of asubstrate. A substrate may be selected from is selected from the groupconsisting of four-helix bundle factors, EGF-like factors, insulin-likefactors, β-trefoil factors and cysteine knot factors. In certainembodiments, the substrate is a receptor, particularly a receptor withsome portion exposed to the extracellular surface. Optionally, thesubstrate is a unique receptor subunit of a heteromeric receptorcomplex. In certain embodiments, the biomolecule is a component of abiomolecular accretion, such as an amyloid deposit or an atheroscleroticplaque. In certain embodiments, the substrate is an intracellularbiomolecule, and in such instances, it may be desirable to use an adzymethat is able to enter the targeted cells, such as an adzyme that furthercomprises a transcytosis moiety that promotes transcytosis of the adzymeinto the cell. In certain embodiments, the substrate is a biomoleculeproduced by a pathogen, such as a protozoan, a fungus, a bacterium or avirus. The substrate may be a prion protein. In a preferred embodiment,the substrate is endogenous to a human patient. In such an embodiment,the adzyme is preferably effective against the substrate in the presenceof physiological levels of an abundant human serum protein, such as,serum albumins or an abundant globin.

[0055] In a preferred embodiment, the substrate for an adzyme is TNFα.In the case of a direct adzyme, the targeting moiety binds to TNFα.Preferably, the catalytic domain comprises a protease that decreasesTNFα activity. For example, the protease is may be selected from among:MT1-MMP; MMP12; tryptase; MT2-MMP; elastase; MMP7; chymotrypsin; andtrypsin. The targeting moiety may be selected from among, a solubleportion of a TNFα receptor and a single chain antibody that binds toTNFα, although other targeting moieties are possible. A preferredtargeting moiety is an sp55 portion of TNFβ Receptor I (TNFR1).

[0056] In another preferred embodiment, the substrate for an adzyme isan interleukin-1, such as IL-1α or IL-1β. In the case of a directadzyme, the targeting moiety binds to the interleukin-1 substrate.Preferably, the catalytic domain comprises a protease that decreases anIL-1 bioactivity.

[0057] In one aspect, the invention provides adzymes for enzymaticallyaltering a substrate, the adzyme comprising: a catalytic domain thatcleaves at least one peptide bond of said substrate to produce one ormore products, and a polypeptide targeting domain that reversibly bindswith an address site on said substrate or with an address site on asecond molecule that occurs in functional proximity to the substrate,wherein said adzyme is resistant to cleavage by the catalytic domain,said targeting moiety, when provided separately, binds to the substrate,said catalytic domain, when provided separately, cleaves at least onepeptide bond of said substrate to produce one or more products, and saidchimeric protein construct is more potent than said catalytic domain ortargeting moiety with respect to the reaction with said substrate.

[0058] In certain embodiments of a proteolytic adzyme, a catalyticdomain and a targeting domain of the adzyme are joined by a polypeptideand linker to form a fusion protein. A fusion protein may be generatedin a variety of ways, including chemical coupling and cotranslation. Ina preferred embodiment, the fusion protein is a cotranslational fusionprotein encoded by a recombinant nucleic acid. In certain embodimentsthe linker for the fusion protein is an unstructured peptide.Optionally, the linker includes one or more repeats of Ser₄Gly orSerGly₄. In preferred embodiments, the linker is selected to providesteric geometry between said catalytic domain and said targeting moietysuch that said adzyme is more effective against the substrate thaneither the catalytic domain or targeting moiety alone. For example, thelinker may be selected such that the adzyme is more potent than saidcatalytic domain or targeting moiety with respect to the reaction withsaid substrate. The linker may be selected such that the targetingmoiety presents the substrate to the enzymatic domain at an effectiveconcentration at least 5 fold greater than would be present in theabsence of the targeting moiety.

[0059] In certain embodiments of a proteolytic adzyme, the adzyme is animmunoglobulin fusion, wherein the catalytic domain and the targetingmoiety are joined, in a geometry consistent with effectiveness againstsubstrate, to at least a portion of an immunoglobulin comprising aconstant domain of an immunoglobulin. For example, the adzyme maycomprise a first fusion protein and a second fusion protein, wherein thefirst fusion protein comprises a constant portion of an immunoglobulinheavy chain and a catalytic domain, and wherein the second fusionprotein comprises a constant portion of an immunoglobulin heavy chainand a targeting domain that reversibly binds with an address site on orin functional prosimity to the substrate. Preferably the immunoglobulinportions are Fc portions that dimerize by disulfide bonds.

[0060] In certain embodiments of a proteolytic adzyme, the adzyme isdesigned so as to have one or more desirable properties adzyme has oneor more desirable properties, with respect to the reaction with saidsubstrate. In many instances, such properties will be significant forachieving the desired effect of the adzyme on the substrate. Forexample, an adzyme may have a potency at least 2 times greater than thecatalytic domain or the targeting moiety alone, and preferably at least3, 5, 10, 20 or more times greater than the potency of the catalyticdomain or targeting moiety alone. The adzyme may have a k_(on) of 10³M⁻¹s⁻¹ or greater, and optionally a k_(on) of 10⁴ M⁻¹s⁻¹, 10⁵ M⁻¹s⁻¹,10⁶ M⁻¹s⁻¹, 10⁷ M⁻¹s⁻¹ or greater. The adzyme may have a k_(cat) of 0.1sec⁻¹ or greater, and optionally a k_(cat) of 1 sec⁻¹, 10 sec⁻¹, 50sec⁻¹ or greater. The adzyme may have a K_(D) that is at least 5, 10,25, 50 or 100 or more fold less than the K_(m) of the catalytic domain.The adzyme may have a k_(off) of 10⁻⁴ sec⁻¹ or greater, and optionally ak_(off) of 10⁻² sec⁻¹, k_(off) of 10⁻² sec⁻¹, or greater. The adzyme mayhave a catalytic efficiency that is at least 5 fold greater than thecatalytic efficiency of the catalytic domain alone, and optionally acatalytic efficiency that is at least 10 fold, 20 fold, 50 fold or 100fold greater than that of the catalytic domain. The adzyme may have aK_(m) at least 5 fold, 10 fold, 20 fold, 50 fold, or 100 fold less thanthe Km of the catalytic domain alone. The adzyme may have an effectivesubstrate concentration that is at least 5 fold, 10 fold, 20 fold, 50fold or 100 fold greater than the actual substrate concentration. Incertain preferred embodiments, an adzyme will be designed so as tocombine two or more of the above described properties.

[0061] In certain embodiments of the proteolytic adzyme, the substrateis a polypeptide produced by a cell. In certain embodiments, thesubstrate is a diffusible extracellular polypeptide, and preferably anextracellular polypeptide signaling molecule that may act on anextracellular or intracellular receptor to triggers receptor-mediatedcellular signaling. Optionally, the substrate is an inflammatorycytokine. In a preferred embodiment, the substrate is an interleukin-1(e.g., IL-1α, IL-1β) or a TNF-α. In certain embodiments, the substrateis a polypeptide hormone, a growth factor and/or a cytokine, especiallyan inflammatory cytokine. Optionally, the adzyme acts to reduces aproinflammatory activity of a substrate. A substrate may be selectedfrom is selected from the group consisting of four-helix bundle factors,EGF-like factors, insulin-like factors, β-trefoil factors and cysteineknot factors. In certain embodiments, the substrate is a receptor,particularly a receptor with some portion exposed to the extracellularsurface. Optionally, the substrate is a unique receptor subunit of aheteromeric receptor complex. In certain embodiments, the biomolecule isa component of a biomolecular accretion, such as an amyloid deposit oran atherosclerotic plaque. In certain embodiments, the substrate is anintracellular biomolecule, and in such instances, it may be desirable touse an adzyme that is able to enter the targeted cells, such as anadzyme that further comprises a transcytosis moiety that promotestranscytosis of the adzyme into the cell. In certain embodiments, thesubstrate is a biomolecule produced by a pathogen, such as a protozoan,a fungus, a bacterium or a virus. The substrate may be a prion protein.In a preferred embodiment, the substrate is endogenous to a humanpatient. In such an embodiment, the adzyme is preferably effectiveagainst the substrate in the presence of physiological levels of anabundant human serum protein, such as, serum albumins or an abundantglobin.

[0062] In general, with a proteolytic adzyme, it will be desirable todesign the adzyme such that it is resistant to cleavage by the proteasecatalytic domain. The protease domain may be generated as a zymogen (aninactive form) and then activated prior to use. The adzyme may bepurified from a cell culture in the presence of a reversible proteaseinhibitor, and such inhibitor may be included in any subsequentprocessing or storage activities.

[0063] In certain embodiments of a proteolytic adzyme, a targetingmoiety may include essentially any molecule or assembly of moleculesthat binds to the address site (e.g., on the substrate in the case ofdirect adzymes or on a molecule that occurs in functional proximity tothe substrate, in the case of proximity adzymes). In many embodiments, atargeting moiety will comprise a polypeptide or polypeptide complex, andparticularly an antibody or polypeptide(s) including an antigen bindingsite of an antibody. For example, a targeting moiety may include amonoclonal antibody, an Fab and F(ab)2, an scFv, a heavy chain variableregion and a light chain variable region. Optionally, the targetingmoiety is an artificial protein or peptide sequence engineered to bindto the substrate. In certain embodiments, the targeting moiety is apolyanionic or polycationic binding agent. Optionally, the targetingmoiety is an oligonucleotide, a polysaccharide or a lectin. In certainembodiments, the substrate is a receptor, and the targeting moietyincludes a ligand (or binding portion thereof) that binds to thereceptor. In certain embodiments, the substrate is a ligand of areceptor, and the targeting moiety includes a ligand binding portion ofthe receptor, particularly a soluble ligand binding portion.

[0064] In a preferred embodiment, the substrate for an adzyme is TNFα.In the case of a direct adzyme, the targeting moiety binds to TNFα.Preferably, the catalytic domain comprises a protease that decreasesTNFα activity. For example, the protease is may be selected from among:MT1-MMP; MMP12; tryptase; MT2-MMP; elastase; MMP7; chymotrypsin; andtrypsin. The targeting moiety may be selected from among, a solubleportion of a TNFα receptor and a single chain antibody that binds toTNFα, although other targeting moieties are possible. A preferredtargeting moiety is an sp55 portion of TNFα Receptor 1 (TNFR1).

[0065] In another preferred embodiment, the substrate for an adzyme isan interleukin-1, such as IL-1α or IL-1β. In the case of a directadzyme, the targeting moiety binds to the interleukin-1 substrate.Preferably, the catalytic domain comprises a protease that decreases anIL-1 bioactivity.

[0066] In one aspect, the invention provides adzymes for enzymaticallyaltering a substrate, the adzyme comprising a polypeptide comprising: acatalytic domain that catalyzes a chemical reaction converting saidsubstrate to one or more products, a targeting domain that reversiblybinds with an address site on said substrate or with an address site ona second molecule that occurs in functional proximity to the substrate,and a linker joining said catalytic domain and said targeting domain,wherein said substrate is an extracellular polypeptide signalingmolecule, said targeting moiety and said catalytic domain areheterologous with respect to each other, said targeting domain, whenprovided separately, binds to said substrate, said catalytic domain,when provided separately, catalyzes the chemical reaction convertingsaid substrate to one or more products, and said adzyme is more potentthan said catalytic domain or targeting moiety with respect to thereaction with said substrate.

[0067] In certain aspects, the invention provides an adzyme forinhibiting receptor-mediated signaling activity of an extracellularsubstrate polypeptide, the adzyme being a fusion protein comprising aprotease domain that catalyzes the proteolytic cleavage of at least onepeptide bond of the substrate polypeptide so as to inhibit thereceptor-mediated signaling activity of the polypeptide, and a targetingdomain that reversibly binds with an address site on said substratepolypeptide, wherein said targeting domain and said protease domain arediscrete and heterologous with respect to each other. Optionally, theadzyme is resistant to cleavage by said protease domain. Optionally, theprotease domain is a zymogen. Optionally, the protease domain isselected from among: a serine proteinase, a cysteine protease, athreonine protease, an aspartate protease and a metalloproteinase.Optionally, the adzyme is purified from a cell culture in the presenceof a reversible protease inhibitor that inhibits the protease activityof the protease domain. In certain embodiments, the adzyme has one ormore properties, with respect to the reaction with said substrate Inmany instances, such properties will be significant for achieving thedesired effect of the adzyme on the substrate. For example, an adzymemay have a potency at least 2 times greater than the catalytic domain orthe targeting moietv alone, and preferably at least 3, 5, 10, 20 or moretimes greater than the potency of the catalytic domain or targetingmoiety alone. The adzyme may have a k_(on) of 10³ M⁻¹s⁻¹ or greater, andoptionally a k_(on) of 10⁴ M⁻¹s⁻, 10⁵ M⁻¹s⁻¹, 10⁶ M⁻¹s⁻¹, 10⁷ M⁻¹s⁻¹ orgreater. The adzyme may have a k_(cat) of 0.1 sec⁻¹ or greater, andoptionally a k_(cat) of 1 sec⁻¹, 10 sec⁻¹, 50 sec¹ or greater. Theadzyme may have a K_(D) that is at least 5, 10, 25, 50 or 100 or morefold less than the K_(m) of the catalytic domain. The adzyme may have ak_(off) of 10⁴ sec⁻¹ or greater, and optionally a k_(off) of 10⁻² sec⁻¹,k_(off) of 10⁻² sec¹, or greater. The adzyme may have a catalyticefficiency that is at least 5 fold greater than the catalytic efficiencyof the catalytic domain alone, and optionally a catalytic efficiencythat is at least 10 fold, 20 fold, 50 fold or 100 fold greater than thatof the catalytic domain. The adzyme may have a Km at least 5 fold, 10fold, 20 fold, 50 fold, or 100 fold less than the K_(m) of the catalyticdomain alone. The adzyme may have an effective substrate concentrationthat is at least 5 fold, 10 fold, 20 fold, 50 fold or 100 fold greaterthan the actual substrate concentration. In certain preferredembodiments, an adzyme will be designed so as to combine two or more ofthe above described properties. Optionally, the substrate is aninflammatory cytokine. In a preferred embodiment, the substrate is aninterleukin-1 (e.g., IL-1α, IL-1β) or a TNF-α. In certain embodiments,the substrate is a polypeptide hormone, a growth factor and/or acytokine, especially an inflammatory cytokine. Optionally, the adzymeacts to reduces a pro-inflammatory activity of a substrate. A substratemay be selected from is selected from the group consisting of four-helixbundle factors, EGF-like factors, insulin-like factors, β-trefoilfactors and cysteine knot factors. In a preferred embodiment, thesubstrate is endogenous to a human patient. In such an embodiment, theadzyme is preferably effective against the substrate in the presence ofphysiological levels of an abundant human serum protein, such as, serumalbumins or an abundant globin. The fusion protein adzymes may begenerated in a variety of ways, including chemical coupling andcotranslation. In a preferred embodiment, the fusion protein is acotranslational fusion protein encoded by a recombinant nucleic acid. Incertain embodiments the linker for the fusion protein is an unstructuredpeptide. Optionally, the linker includes one or more repeats of Ser₄Glyor SerGly₄. In preferred embodiments, the linker is selected to providesteric geometry between said catalytic domain and said targeting moietysuch that said adzyme is more effective against the substrate thaneither the catalytic domain or targeting moiety alone. For example, thelinker may be selected such that the adzyme is more potent than saidcatalytic domain or targeting moiety with respect to the reaction withsaid substrate. The linker may be selected such that the targetingmoiety presents the substrate to the enzymatic domain at an effectiveconcentration at least 5 fold greater than would be present in theabsence of the targeting moiety. A targeting domain may includeessentially any molecule or assembly of molecules that binds to theaddress site (e.g., on the substrate in the case of direct adzvmes or ona molecule that occurs in functional proximity to the substrate, in thecase of proximity adzymes). In many embodiments, a targeting domain willcomprise an antigen binding site of an antibody, such as a single chainantibody. Optionally, the targeting moiety is an artificial protein orpeptide sequence engineered to bind to the substrate. In a preferredembodiment, the substrate for the adzyme is TNFα. In the case of adirect adzyme, the targeting moiety binds to TNFα. Preferably, thecatalytic domain comprises a protease that decreases TNFα activity. Forexample, the protease is may be selected from among: MT1-MMP; MMP12;tryptase; MT2-MMP; elastase; MMP7; chymotrypsin; and trypsin. Thetargeting moiety may be selected from among, a soluble portion of a TNFareceptor and a single chain antibody that binds to TNFα, although othertargeting moieties are possible. A preferred targeting moiety is an sp55portion of TNFα Receptor 1 (TNFR1). In another preferred embodiment, thesubstrate for the adzyme is an interleukin-1, such as IL-1α or IL-1β. Inthe case of a direct adzyme, the targeting moiety binds to theinterleukin-1 substrate. Preferably, the catalytic domain comprises aprotease that decreases an IL-1 bioactivity.

[0068] In one aspect, the invention provides an adzyme for inhibitingreceptor-mediated signaling activity of an extracellular substratepolypeptide, the adzyme being an immunoglobulin fusion complex. Forexample, such an adzyme may comprise: a first fusion protein bound to asecond fusion protein, wherein the first fusion protein comprises aconstant portion of an immunoglobulin heavy chain and a protease domainthat catalyzes the proteolytic cleavage of at least one peptide bond ofthe substrate polypeptide so as to inhibit the receptor-mediatedsignaling activity of the polypeptide, and wherein the second fusionprotein comprises a constant portion of an immunoglobulin heavy chainand a targeting domain that reversibly binds with an address site onsaid substrate polypeptide, wherein said targeting domain and saidprotease domain are discrete and heterologous with respect to eachother.

[0069] In certain aspects, the invention provides adzyme preparationsfor use in a desired application, such as a therapeutic use, anindustrial use, an environmental use or in a microfabrication. Suchpreparations may be termed adzyme preparations. In certain embodiments,the invention provides an adzyme preparation for therapeutic use in ahuman patient, the preparation comprising any adzyme disclosed herein.Optionally, the preparation further comprising a pharmaceuticallyeffective carrier. Optionally, the adzyme preparation is formulated suchthat autocatalytic modification of the adzyme is inhibited. Optionally,the adzyme comprises a catalytic domain that is a protease, and incertain embodiments, the preparation comprises a reversible inhibitor ofsaid protease, preferably a reversible inhibitor that is safe foradministration to a human patient. Optionally, an adzyme preparation fortherapeutic use is substantially pyrogen free. An adzyme preparation maybe packaged along with instructions for use. For example, an adzymepreparation for therapeutic use may be packaged with instructions foradministration to a patient.

[0070] In certain aspects, the invention provides methods for making amedicament for use in treating a disorder that is associated with anactivity of the substrate of any adzyme disclosed herein, the methodcomprising formulating the adzyme for administration to a patient,preferably a human patient. In certain embodiments, the inventionprovides a method of making a medicament for use in treating aninflammatory or allergic disorder, the method comprising formulating anadzyme for administration to a human patient in need thereof, whereinthe substrate of the adzyme is an inflammatory cytokine.

[0071] In certain aspects, the invention provides methods of treating adisorder that is associated with an activity of the substrate of anadzyme, the method comprising administering a therapeutically effectivedose of an adzyme to a human patient in need thereof. In certainembodiments, an adzyme may be used in a method of treating aninflammatory of allergic disorder, the method comprising administering atherapeutically effective dose of an adzyme to a human patient in needthereof, wherein the substrate of the adzyme is an inflammatorycytokine.

[0072] In certain aspects, the invention provides nucleic acids encodingany of the various polypeptide portions of an adzyme, and particularlyrecombinant nucleic acids encoding a fusion protein adzyme. Such nucleicacids may be incorporated into an expression vector wherein theexpression vector directs expression of the adzyme in a suitable hostcell. The invention further provides cells comprising such nucleic acidsand vectors. In certain embodiments, the invention provides cellscomprising a first nucleic acid comprising a first coding sequence and asecond nucleic acid comprising a second coding sequence, wherein thefirst coding sequence encodes a first fusion protein comprising animmunoglobulin heavy chain and a catalytic domain, and wherein thesecond coding sequence encodes a second fusion protein comprising animmunoglobulin heavy chain and a targeting domain. Preferably, such ascell, in appropriate culture conditions, secretes an adzyme comprisingan Fc fusion protein construct that is a dimer of the first fusionprotein and the second fusion protein.

[0073] In certain aspects, the invention provides methods formanufacturing an adzyme. Such methods may include expression ofpolypeptide components in cells. Such methods may include chemicaljoining of various adzyme components. In one embodiment, a methodcomprises culturing a cell having an expression vector for producing afusion protein adzyme in conditions that cause the cell to produce theadzyme encoded by the expression vector; and purifying the adzyme tosubstantial purity. In one embodiments, a method comprises culturing acell designed to produce an immunoglobulin fusion in conditions thatcause the cell to produce the adzyme encoded by the expression vector;and purifying the adzyme to substantial purity. In certain embodiments,purifying an adzyme to substantial purity includes the use of areversible inhibitor that inhibits autocatalytic activity of thecatalytic domain, and particularly, wherein the catalytic domain of theadzyme is a protease domain, and wherein purifying the adzyme tosubstantial purity includes the use of a reversible protease inhibitorthat inhibits the protease activity of the catalytic domain.

[0074] In further aspects, the invention provides methods for designingand producing adzymes with desirable properties, and methods foroperating a business that involves designing and selling adzymes withdesirable properties, such as therapeutically effective adzymes.

[0075] The embodiments and practices of the present invention, otherembodiments, and their features and characteristics, will be apparentfrom the description, figures and claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0076] FIGS. 1A-1J are schematic representations of the structure of aseries of different exemplary constructs embodying the invention. Theboxes represent moieties having binding or catalytic properties, and canbe embodied as true protein domains, i.e., bonded sequences of aminoacids forming structures characterized by folding of the peptide chaininto alpha helices, beta pleated sheets, random coils, etc., to formseparate binding surfaces or enzymatically active sites, and includingcatalytic moieties (CAT), address moieties (ADD), and protein domainsserving to associate these parts together in various operativeconfigurations. Lines connecting boxes represent a covalent bond linkingtogether amino acid sequence defining the respective functional regions,or linkers comprising, for example, a flexible linear linker such as astring of peptide bonded amino acids or a poly(ethylene glycol) chain.Lines between boxes represent non covalent, reversible attachmentswherein the parts are held together by a combination of forces such ashydrogen bonding, hydrophobic-hydrophobic interaction, opposite chargematching, etc., for example, ligand-receptor interactions.

[0077]FIG. 1K is a schematic diagram illustrating the basic concept of acontingent adzyme.

[0078] FIGS. 2A-2J are cartoons illustrating various exemplaryembodiments of adzyme constructs at various types of targetedbiomolecules in position to initiate an enzymatic reaction on thesubstrate site of the target. The address is designated as AD, thecatalytic domain as CD.

[0079] FIGS. 3A-3G are cartoons illustrating various exemplaryembodiments of contingent adzyme constructs in the absence of and in thevicinity of their respective intended targeted biomolecules.

[0080]FIG. 4 is a cartoon illustrating components of a pre thrombinscFvαHA adzyme.

[0081]FIG. 5 is electrophoretic analysis of purified model adzyme.

[0082]FIG. 6 is Western blot analysis of model adzyme activated usingFactor Xa.

[0083]FIG. 7 shows proteolytic activity of thrombin and model adzymebefore and after activation on standard thrombin tripeptide substrate.

[0084]FIG. 8 shows that enhanced adzyme activity is driven by thepresence of an address domain.

[0085]FIG. 9 shows that enhanced adzyme activity requirescotranslational linkage of the domains.

[0086]FIG. 10 shows proteolytic inactivation of TNFα cytotoxicity.

[0087]FIG. 11 shows that soluble TNFα receptor p55 address domain bindsTNFα.

[0088]FIG. 12 is a representative expression of several adzymeconstrcuts as analyzed by Western blotting with anti-myc antibody. Lane1: trypsinogen expressed in the absence of stabilizing benzamidine, Lane2: trypsinogen, Lane 3: trypsinogen-0aa-sp55, Lane 4:trypsinogen-20aa-sp55; Lane 5: trypsinogen-3aa-sp55, Lane 6: sp55.Material in lanes 2 through 6 was expressed in the presence of 1 mMbenzamidine.

[0089]FIG. 13 shows a snapshot of representative experiments where thefluorescence detected at the end of 2 hours of incubation is comparedfor the different recombinant adzymes and other control proteins.

[0090]FIG. 14 shows normalization of trypsin activities.

[0091]FIG. 15 shows detection of TNFα binding of adzymes by ELISA.

[0092]FIG. 16 shows kinetic model results comparing the performance ofan adzyme, an address, and an enzyme.

DETAILED DESCRIPTION OF THE INVENTION

[0093] I. Overview

[0094] The invention provides a new class of engineered proteinconstructs, referred to herein as “adzymes”, as well as methods andcompositions related to the use and production of adzymes. Adzymes arechimeric protein constructs that join one or more catalytic domains withone or more targeting moieties (or “addresses”). A catalytic domain ofan adzyme has an enzymatically active site that catalyzes a reactionconverting a pre-selected substrate (the “target” or “targetedsubstrate”) into one or more products, such as by cleavage, chemicalmodifications (transformations) or isomerization. Generally, thecatalytic domain is selected such that one or more of the product(s) ofthe adzyme-mediated reaction have a qualitatively or quantitativelydifferent activity relative to the selected substrate. Merely toillustrate, the adzyme may alter such functional characteristics of aselected substrate as affinity, potency, selectivity, solubility,immunogenicity, half-life, clearance (such as by renal or hepaticfunction), biodistribution or other pharmacokinetic properties. Incertain instances, the product of an adzyme-mediated reaction is itselfan antagonist of an activity of the selected substrate.

[0095] The targeting moiety (or “address”) is a moiety capable ofrecognizing and reversibly binding to a pre-determined “address bindingsite” (also herein “address site”), such as, for example, a soluble ormembrane-bound biomolecules, or a component of a biomolecular accretion(e.g., a plaque or other insoluble protein-containing aggregate). Incertain types of adzymes (termed “direct adzymes”), the targeting moietybinds to the target molecule. In certain types of adzymes (termed“proximity adzymes”) the targeting moiety binds to a molecule that tendsto occur in functional proximity to the target. The term “moiety” shouldbe understood as including single molecules or portions thereof (e.g., apolypeptide or sugar that binds to the address binding site), as well ascombinations of molecules (e.g., an antibody that binds to an addressbinding site).

[0096] In an adzyme, at least one targeting moiety is operativelyassociated with at least one catalytic domain. An adzyme may be a singlepolypeptide chain (e.g., a fusion protein) or an assembly of polypeptidechains and/or other molecules that are joined through covalent ornon-covalent bonds. Regardless of how the constituent portions of anadzyme are associated, at least one targeting moiety and one catalyticdomain should be operatively associated. The term “operativelyassociated”, as used herein to describe the relationship between acatalytic domain and a targeting moiety, means that the effectiveness ofthe associated catalytic domain and targeting moiety in chemicallyaltering or otherwise affecting the activity of the pre-selectedsubstrate is greater than the effectiveness of either the targetingmoiety or the catalytic domain alone, and also greater than theeffectiveness of both the targeting moiety and the catalytic domain whenprovided in combination but not in association with each other (e.g.,where the target is simultaneously contacted with both a discretecatalytic domain and a discrete targeting moiety). As described below,the adzyme may include other components as well, such as linkers,moieties that influence stability or biodistribution, and the like.

[0097] The effectiveness of an adzyme relative to its constituent partsmay be assessed in a variety of ways. For example, effectiveness may beassessed in terms of potency of the adzyme, as compared to its componentparts, to affect a biological activity of the pre-selected substrate. Asanother example, effectiveness may be assessed in terms of a comparisonof kinetic or equilibrium constants that describe the reaction betweenthe adzyme and the pre-selected substrate to those that apply to thereaction between the component parts and the targeted substrate. Inembodiments where an adzyme is intended for use in a mammal, at leastone catalytic domain and at least one targeting moiety of an adzyme willbe associated such that these portions are operatively associated underphysiological conditions (e.g., in whole blood, serum, cell cultureconditions, or phosphate buffered saline solution, pH 7). Where theadzyme is intended for other purposes (e.g., the modification of anenvironmental pollutant or the modification of a component of amolecular reaction), at least one catalytic domain and at least onetargeting moiety of an adzyme will be associated such that theseportions are operatively associated under the expected or desiredreaction conditions.

[0098] Merely to illustrate, an adzyme may comprise a catalytic domainthat cleaves or otherwise modifies TNF-α, converting it into one or moreproducts having reduced activity, no activity or antagonist activity,thereby ameliorating a disease state associated with TNF-α, such asrheumatoid arthritis or other conditions associated with TNF-α activity.

[0099] While not wishing to be bound to any particular mechanism ofaction, it is expected that a targeting moiety will bind to thepre-selected targeted substrate (direct adzyme) or to another moleculethat occurs in the same vicinity as the pre-selected targeted substrate(proximity adzyme), and thereby functions to increase the concentrationof the catalytic domain at or near the targeted substrate. In this way,the adzyme is self-concentrating at or in the vicinity of a targetedsubstrate and has an enhanced effectiveness for reacting with andaltering the activity of the targeted substrate, relative to thecatalytic or binding domains alone. As a consequence to the improvedeffectiveness of the targeted reaction, the adzyme has a greaterselectivity and/or catalytic efficiency for the targeted substrate ascompared to other non-targeted (potential) substrates of the catalyticdomain.

[0100] Again, while not wishing to be bound to any particular theory,for certain adzymes it is expected that a relatively fast k_(on) ratefor the targeted substrate will be desirable. A k_(on) of at least 10³M⁻¹s⁻¹ may be desirable. Other kinetic and performace parameters thatmay be useful in certain embodiments are described below.

[0101] In most embodiments, the modular components of an adzyme areheterologous with respect to each other, meaning that these domains arenot found naturally as part of a single molecule or assembly ofmolecules, and accordingly, adzymes of these embodiments are notnaturally occurring substances. Each of the various domains and moietiesthat are present in an adzyme may themselves be a naturally occurringprotein or protein fragment, or other naturally occurring biomolecule(e.g., a sugar, lipid or non-proteinaceous factor), or an engineered orwholly synthetic molecule.

[0102] In most embodiments, a catalytic domain will comprise apolypeptide having enzymatic activity. In certain preferred embodiments,a targeting moiety will comprise a polypeptide. In general, at least onecatalytic domain and at least one targeting moiety of the adzyme areselected from amongst “modular” entities, i.e., able to function as acatalyst or binding agent independently. To exemplify, an adzyme may bea single fusion protein comprising (1) a catalytic domain that comprisesa polypeptide and has enzymatic activity and (2) an targeting domainthat comprises a polypeptide and binds to an address binding site, and,optionally, (3) a polypeptide linker configured such that the catalyticdomain and targeting domain are operatively associated. As anotherexample, an adzyme may be a type of immunoglobulin fusion construct,wherein a first fusion protein comprises a catalytic domain fused to afirst Fc chain and a second fusion protein comprises a targeting domainfused to a second Fc chain, and wherein the first and second Fc chainsare associated in such a way as to cause the catalytic domain and thetargeting domain to be operatively associated.

[0103] Within the broad category of adzymes, various subcategories orclasses of adzymes may be identified. As noted above, two such classesare termed herein “direct” adzymes and “proximity” adzymes. In a directadzyme the targeting moiety binds to a targeted substrate. The catalyticdomain acts on the same type of molecule as the targeting moiety hasbound. In certain embodiments, this will require the targeting moiety todissociate from the targeted substrate in order for the catalytic domainto alter that molecule. Depending on a variety of conditions, such asthe concentration of the direct adzyme and the concentration of thetargeted substrate, the catalytic domain of a direct adzyme mayprimarily act on the targeted substrate that is or was bound by thetargeting moiety, or the direct adzyme may act on one substrate whilethe targeting moiety is bound to another. While not wishing to be boundto mechanism, it is generally expected that when the targeted substrateis present in relatively low concentrations (as is the case for mostextracellular signaling molecules in the extracellular fluids of amulticellular organism), a direct adzyme will primarily act on thetargeted substrate that is or was bound by the targeting moiety. In aproximity adzyme, the targeting moiety binds to a molecule that is notcovalently part of the targeted substrate. Instead, the targeting moietybinds to a molecule that is expected to be found in functional proximityto the targeted substrate. By “functional proximitv” is meant that theaddress binding site is present at sufficient concentration or withsufficient stability in the proximity of targeted substrates that theadzyme reacts with the targeted substrate with greater effectivenessthan the catalytic domain and targeting moiety alone or innon-associated combination. While the existence of functional proximitybetween an address binding site and a targeted substrate is mostaccurately assessed in the milieu in which the adzyme is intended foruse (e.g., in the human body, in a contaminated soil site), an adzymemay be considered a proximity adzyme if it shows the appropriateeffectiveness in a reasonable experimental system, such as a culture ofcells related to the type of cells that are predicted to be targeted bythe adzyme, or in a purified protein mixture where the address bindingsite and the adzyme are present at concentrations that fairlyapproximate those that are expected in the intended milieu. In certainembodiments, the targeting moiety binds to a molecule which isdiffusionally constrained with respect to the targeted substrate,meaning that, for whatever reason, the targeted substrate and theaddress binding site are neither covalently attached nor free to diffuseapart. For example, the targeting moiety may bind one protein in areceptor complex while the catalytic domain acts on another protein inthe receptor complex. As another example, the targeting moiety may bindto a protein that is lodged in cell membranes and the targeted substratemay also be lodged in or attached to cell membranes. The terms “directadzyme” and “indirect a dzyme”, while distinct concepts that raisedifferent issues in adzyme design, may not, in practice, be entirelymutually exclusive. For example, an targeting moiety may bind to boththe targeted substrate and a separate molecule that occurs in functionalproximity to the targeted substrate.

[0104] An additional discernible class of adzymes are the “contingentadzymes”. The term “contingent adzymes” refers to adzyme constructs thatare catalytically activated or upregulated in the vicinity of thetargeted substrate but less active, such as by inhibition, elsewhere.Both direct and proximity adzymes can be modified to be contingentadzymes, in which the interaction of the targeting domain with itscognate partner alters the activity of the catalytic domain, such as byallosteric, competitive, or non-competitive mechanisms.

[0105] As a descriptive example, a variety of antibodies with affinityfor particular targets (e.g., anti-TNF-α and anti-EGF receptor) havebeen used as effective therapeutic agents for certain disorders, and itis expected, in accordance with the teachings herein, that adzymes withgreater potency than the antibodies alone may be designed.

[0106] In a further aspect, the present invention providespharmaceutical compositions comprising an adzyme of the invention and apharmaceutically acceptable carrier, as well as methods for making amedicament for use in a human by combining an adzyme with apharmaceutically acceptable carrier.

[0107] In another aspect, the present invention provides a method fortreating a subject, e.g., a human, suffering from a disease. The methodincludes administering (e.g., using a pharmaceutical formulation) atherapeutically, prophylactically or analgesically effective amount ofan adzyme, thereby treating a subject suffering from a disease. In oneembodiment, the disease is associated with a soluble molecule and theadzyme is administered to the subject in an amount effective to renderthe soluble molecule biologically inactive.

[0108] II. Definitions

[0109] For convenience, certain terms employed in the specification,examples, and appended claims are collected here. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

[0110] As used herein, the term “aptamer”, when referring to a targetingmoiety, encompasses an oligonucleotide that interacts with a targetedsubstrate or associated molecule, e.g., binds to the address site for anadzyme.

[0111] As used herein, the term “biologically inactive” as it relates toa targeted biomolecule is intended to mean that its biological functionis down-regulated, e.g., suppressed or eliminated. For example, if thetarget is TNFα, biological inactivation would include modifying TNFαsuch that the inflammatory response mediated by NFKB is inhibited, thereis inhibition of the secretion of other pro-inflammatory cytokines, theinduction of endothelial procoagulant activity of the TNF is inhibited;the binding of TNF to receptors on endothelial cells is inhibited; theinduction of fibrin deposition in the tumor and tumor regressionactivities of the TNF are enhanced; and/or the cytotoxicity and receptorbinding activities of the TNF are unaffected or enhanced on tumor cells.For example, a catalytic domain capable of methylating TNFα (e.g.,methylating TNFα on ¹⁵His as described in Yamamoto R. et al. (1989)Protein Engineering 2(7):553-8) would inactivate TNFα.

[0112] The term “k_(cat)”, or the “turnover number”, is the number ofsubstrates converted to product per enzyme molecule per unit of time,when E is saturated with substrate.

[0113] The term “k_(cat)/K_(m)”, is an apparent second-order rateconstant that is a measure of how the enzyme performs when theconcentration of substrate is low (e.g., not saturating). The upperlimit for k_(cat)/K_(m) is the diffusion limit—i.e., the rate at whichenzyme and substrate diffuse together. k_(cat)/K_(m) is also known asthe “catalytic efficiency” for the enzyme.

[0114] The term “catalytic efficiency”, as applied to an adzyme, is theapparent second-order rate constant of the adzyme when the concentrationof substrate is substantially (at least ten-fold) lower than theMichaelis-Menten constant (Km) for the adzyme (i.e., when [S]<<K_(m)),at least with respect to those adzymes that can be reasonably modeledusing Michaelis-Menten kinetic modeling theories. In the case of manysimple catalytic domains taken in isolation, the catalytic efficiencymay be defined as the ratio k_(cat)/K_(m) (see above).

[0115] In most cases where Michaelis-Menten modeling applies, thecatalytic efficiency will be different for the adzyme and for itscomponent enzyme, i.e. the adzyme's catalytic efficiency is notk_(cat)/K_(m). Both v_(max) and K_(m) are also different for the adzyme.For a case where the Michaelis-Menten pseudo-steady state analysis isvalid (generally [AE]₀, <<[S]₀, wherein [AE]₀ is the initial adzymeconcentration) and substrate holdup is negligible, simple closed-formexpressions for these quantities can be derived: $\begin{matrix}{v_{\max}^{AE} = {\frac{k_{off}^{AS}}{k_{cat}^{ES} + {k_{off}^{AS}{K_{m}^{E}/\lbrack S\rbrack_{eff}}} + k_{off}^{AS}}v_{\max}^{E}}} \\{K_{m}^{AE} = \frac{( {{k_{off}^{AS}{K_{m}^{E}/\lbrack S\rbrack_{eff}}} + k_{cat}^{ES}} )k_{off}^{AS}}{( {k_{cat}^{ES} + {k_{off}^{AS}{K_{m}^{E}/\lbrack S\rbrack_{eff}}} + k_{off}^{AS}} )k_{on}^{AS}}}\end{matrix}$

[0116] wherein v_(max) ^(AE) and v_(max) ^(E) are the maximum velocityfor the adzyme and its enzyme component, respectively; K_(m) ^(AE) andK_(m) ^(E) are the K_(m) for the adzyme and its enzyme component,respectively. The superscript “AS” indicates that the kinetic constantis that of an address/targeting moiety, which is determined byindependent experiments on the address; the superscript “ES” indicatesthat the kinetic constant is that of an enzyme/catalytic moiety, whichis determined by independent experiments on the enzyme. [S]_(eff) or the“effective concentration” of the targeted substrate is a geometricparameter of the adzyme with concentration units.

[0117] The catalytic efficiency for an adzyme is: $\begin{matrix}{{{Catalytic}\quad {Efficiency}} = \frac{v_{\max}^{AE}}{{K_{m}^{AE}\lbrack{AE}\rbrack}_{o}}} \\{= \frac{k_{on}^{AS}k_{cat}^{ES}}{{k_{off}^{AS}{K_{m}^{E}/\lbrack S\rbrack_{eff}}} + k_{cat}^{ES}}}\end{matrix}$

[0118] A “chimeric protein construct” is an assemblage comprising atleast two heterologous moieties, e.g., a catalytic domain and an addressthat are heterologous with respect to each other, that are covalently ornon-covalently associated to form a complex. A chimeric proteinconstruct may comprise non-proteinaceous molecules.

[0119] “Differentiation” in the present context means the formation ofcells expressing markers known to be associated with cells withdifferent functional properties or cells that are more specialized andcloser to becoming terminally differentiated cells incapable of furtherdivision or differentiation.

[0120] A “fusion protein” is a chimeric protein wherein at least twoheterologous amino acid sequences are covalently joined through an amidebackbone bond, e.g., to form one contiguous polypeptide.

[0121] As used herein, the terms “modulate” or “alter” the activity ofthe targeted substrate are intended to include inhibiting, stimulating,up-regulating, down-regulating, activating, inactivating, or modifyingthe activity of the target in any other way.

[0122] A polynucleotide sequence (DNA, RNA) is “operatively linked” toan expression control sequence when the expression control sequencecontrols and regulates the transcription and translation of thatpolynucleotide sequence. The term “operatively linked” includes havingan appropriate start signal (e.g., ATG) in front of the polynucleotidesequence to be expressed, and maintaining the correct reading frame topermit expression of the polynucleotide sequence under the control ofthe expression control sequence, and production of the desiredpolypeptide encoded by the polynucleotide sequence.

[0123] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention, i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0124] The terms “polynucleotide sequence” and “nucleotide sequence” arealso used interchangeably herein.

[0125] As used herein, “protein” is a polymer consisting essentially ofany of the 20 amino acids. Accordingly, a protein may include variousmodifications (e.g., glycosylation, phosphorylation) or non-amino acids.Although “polypeptide” is often used in reference to relatively largepolypeptides, and “peptide” is often used in reference to smallpolypeptides, usage of these terms in the art overlaps and is varied.

[0126] As used herein, “proliferating” and “proliferation” refer tocells undergoing mitosis.

[0127] The International Union of Biochemistry and Molecular Biology(1984) has recommended to use the term “peptidase” for the subset ofpeptide bond hydrolases (Subclass E. C 3.4.). The widely used termprotease is synonymous with peptidase. Peptidases comprise two groups ofenzymes: the endopeptidases and the exopeptidases. Endopeptidases cleavepeptide bonds at points within a protein, and exopeptidases remove aminoacids sequentially from either the N- or C-terminus.

[0128] The term “proteinase” is also used as a synonym forendopeptidase. Proteinases are classified according to their catalyticmechanisms. Five mechanistic classes have been recognized by theInternational Union of Biochemistry and Molecular Biology: serineproteinases, cysteine proteinases, aspartic proteinases, threonineproteinases, and metalloproteinases.

[0129] This classification by catalytic types has been suggested to beextended by a classification by families based on the evolutionaryrelationships of proteases (Rawlings, N. D. and Barrett, A. J., (1993),Biochem. J., 290, 205-218). This classification is available in theSwissProt database.

[0130] In addition to these five mechanistic classes, there is a sectionof the enzyme nomenclature which is allocated for proteases ofunidentified catalytic mechanism. This indicates that the catalyticmechanism has not been identified, and the possibility remains thatnovel types of proteases do exist.

[0131] The class “serine proteinases” comprises two distinct families:the chymotrypsin family which includes the mammalian enzymes such aschymotrypsin, trypsin or elastase or kallikrein, and the substilisinfamily which includes the bacterial enzymes such as subtilisin. Thegeneral three-dimensional structure is different in the two families butthey have the same active site geometry and catalysis proceeds via thesame mechanism. The serine proteinases exhibit different substratespecificities which are related to amino acid substitutions in thevarious enzyme subsites (see the nomenclature of Schechter and Berger)interacting with the substrate residues. Three residues which form thecatalytic triad are essential in the catalytic process: His-57, Asp-102and Ser-195 (chymotrypsinogen numbering).

[0132] The family of “cysteine proteinases” includes the plant proteasessuch as papain, actinidin or bromelain, several mammalian lysosomalcathepsins, the cytosolic calpains (calcium-activated), and severalparasitic proteases (e.g., Trypanosoma, Schistosoma). Papain is thearchetype and the best studied member of the family. Like the serineproteinases, catalysis proceeds through the formation of a covalentintermediate and involves a cysteine and a histidine residue. Theessential Cys-25 and His-159 (papain numbering) play the same role asSer-195 and His-57 respectively. The nucleophile is a thiolate ionrather than a hydroxyl group. The thiolate ion is stabilized through theformation of an ion pair with neighboring imidazolium group of His-159.The attacking nucleophile is the thiolate-imidazolium ion pair in bothsteps and then a water molecule is not required.

[0133] Most of the “aspartic proteinases” belong to the pepsin family.The pepsin family includes digestive enzymes such as pepsin and chymosinas well as lysosomal cathepsins D, processing enzymes such as renin, andcertain fungal proteases (penicillopepsin, rhizopuspepsin,endothiapepsin). A second family comprises viral proteinases such as theprotease from the AIDS virus (HIV) also called retropepsin. In contrastto serine and cysteine proteinases, catalysis by aspartic proteinasesdoes not involve a covalent intermediate, though a tetrahedralintermediate exists. The nucleophilic attack is achieved by twosimultaneous proton transfers: one from a water molecule to the dyad ofthe two carboxyl groups and a second one from the dyad to the carbonyloxygen of the substrate with the concurrent CO—NH bond cleavage. Thisgeneral acid-base catalysis, which may be called a “push-pull” mechanismleads to the formation of a non-covalent neutral tetrahedralintermediate.

[0134] The “metalloproteinases” are found in bacteria, fungi as well asin higher organisms. They differ widely in their sequences and theirstructures but the great majority of enzymes contain a zinc (Zn) atomwhich is catalytically active. In some cases, zinc may be replaced byanother metal such as cobalt or nickel without loss of the activity.Bacterial thermolysin has been well characterized and itscrystallographic structure indicates that zinc is bound by twohistidines and one glutamic acid. Many enzymes contain the sequenceHEXXH (SEQ ID NO:), which provides two histidine ligands for the zincwhereas the third ligand is either a glutamic acid (thermolysin,neprilysin, alanyl aminopeptidase) or a histidine (astacin). Otherfamilies exhibit a distinct mode of binding of the Zn atom. Thecatalytic mechanism leads to the formation of a non-covalent tetrahedralintermediate after the attack of a zinc-bound water molecule on thecarbonyl group of the scissile bond. This intermediate is furtherdecomposed by transfer of the glutamic acid proton to the leaving group.

[0135] In discussing the interactions of peptides with proteinases,e.g., serine and cysteine proteinases and the like, the presentapplication utilizes the nomenclature of Schechter and Berger [(1967)Biochem. Biophys. Res. Commun. 27:157-162)]. The individual amino acidresidues of a substrate or inhibitor are designated P1, P2, etc. and thecorresponding subsites of the enzyme are designated S1, S2, etc. Thescissile bond of the substrate is P1P1′.

[0136] The binding site for a peptide substrate consists of a series of“specificity subsites” across the surface of the enzyme. The term“specificity subsite” refers to a pocket or other site on the enzymecapable of interacting with a portion of a substrate for the enzyme.

[0137] “Recombinant,” as used herein with respect to a protein, meansthat the protein is derived from the expression of a recombinant nucleicacid by, for example, a prokaryotic, eukaryotic or in vitro expressionsystem. A recombinant nucleic acid is any non-naturally occurringnucleic acid sequence or combination of nucleic acid sequences that wasgenerated as a result of human intervention.

[0138] The term “substrate” refers to a substrate of an enzyme which iscatalytically acted on and chemically converted by the enzyme toproduct(s).

[0139] The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space. In particular, “enantiomers” refer to twostereoisomers of a compound which are non-superimposable mirror imagesof one another. “Diastereomers”, on the other hand, refers tostereoisomers with two or more centers of asymmetry and whose moleculesare not mirror images of one another. With respect to the nomenclatureof a chiral center, terms “D” and “L” configuration are as defined bythe IUPAC Recommendations. As to the use of the terms, diastereomer,racemate, and enantiomer will be used in their normal context todescribe the stereochemistry of peptide preparations.

[0140] “Transcriptional regulatory sequence” is a generic term usedthroughout the specification to refer to nucleic acid sequences, such asinitiation signals, enhancers, and promoters, which induce or controltranscription of protein coding sequences with which they are operablylinked. In some examples, transcription of a recombinant gene is underthe control of a promoter sequence (or other transcriptional regulatorysequence) which controls the expression of the recombinant gene in acell-type in which expression is intended. It will also be understoodthat the recombinant gene can be under the control of transcriptionalregulatory sequences which are the same or which are different fromthose sequences which control transcription of the naturally-occurringform of a protein.

[0141] As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. Preferred vectors are those capable of autonomousreplication and/or expression of nucleic acids to which they are linked.Vectors capable of directing the expression of genes to which they areoperatively linked are referred to herein as “expression vectors”.

[0142] III. Exemplary Embodiments

[0143] An adzyme comprises at least two modular moieties: a targetingmoiety and a catalytic domain. With respect to altering the activity ofa targeted substrate, the adzyme is more potent relative to either thecatalytic domain or targeting moiety alone.

[0144] The catalytic domain will often be protein-based, though eventhen may include other components, such as organic ligands orco-factors, or metal ions. It comprises a catalytically active site thatreacts with a substrate without itself being consumed in the reaction. Acatalytic domain will generally alter one or more bonds of a substrate,e.g., breaking the bond, removing one or more atoms across the bond(including oxidizing or reducing), and/or altering the stereochemistryof an atom participating in the bond. The site of chemical modificationon the targeted substrate is referred to herein as the “substrate site”.

[0145] The targeting moiety recognizes and binds to a pre-determinedmolecule, i.e., an address binding site such as on a soluble or membranebound intracellular or extracellular targeted biomolecule, whichmolecule is the same as or associated with the targeted substrate. Theeffect in both instances is to impart “addressability” to the adzymeconstruct, that is, to increase the local concentration of the constructin the vicinity of the targeted substrate so as to increase theproximity of the catalytic domain to the targeted substrate and therebyincrease the catalytic efficiency for that substrate.

[0146] The targeting moiety and catalytic domain may be covalentlyattached or associated by non-covalent means. For instance, the moietiescan be covalently attached as by fusion of two protein domains, with orwithout intervening sequences, to form a single polypeptide chain, orthrough derivation of the amino or carboxy terminus, or a sidechain of apolypeptide chain. In certain preferred embodiments, the targetingmoiety and catalytic domain are produced as a cotranslational fusion byexpression of a single recombinant nucleic acid construct. The variousmoieties may also be associated by non-covalent interactions, such asbetween protein domains, interaction with a common crosslinking ligand,etc.

[0147] The adzyme concept can be exploited in appropriate circumstancesusing a recruitment approach. Here, a multispecific binder isadministered. An address of the multispecific binder complexes with abinding site on or near the intended targeted biomolecule. A chaperoneprotein or other structure of the multispecific binder, linked to orconstituting a part of the address, displays a surface which complexeswith a catalytic domain such as an enzyme already present in the body,or a co-administered enzymatically active moiety. The multispecificbinder thereby induces complex formation between the address and acatalytic domain. The affinity of the address for the binding siteserves to increase the effective concentration of the catalytic domainin the vicinity of the targeted biomolecule.

[0148] The address and catalytic domain of an adzyme often cooperate toproduce synergistic behavior. The target may be modulated, e.g.,inhibited by cleavage, by a catalytic domain used alone at a potencydetermined by its K_(m) and k_(cat). The target may also be inhibited bybinding with a molecule defining an address used alone at a potencydetermined by its K_(a), acting simply as a conventional drug. Theamount of modulation of the target often may be objectively measured bystandard assays. Thus modulation induced independently through eachmechanism often can be at least roughly quantitated. It often will befound, at least in some adzyme constructs, that an adzyme comprising anoptimized combination of a catalytic domain having the same K_(m) andk_(cat), and an address having the same K_(a) will have a potency atleast 10, 10², 10³, or even 10⁴ times the sum of the potency of theindividual components (catalytic and targeting) acting alone.

[0149] Another way to express the functional improvement of the adzymein a pharmaceutical setting, relative to the targeting moiety and/orcatalytic domain alone, is that in certain preferred embodiments theadzyme will have an effective dose (ED₅₀) for altering the activity ofthe targeted substrate in vivo at least 2 times less than the catalyticdomain and/or targeting moiety (e.g., if a neutralizing moiety) alone,and more preferably at least 5, 10 or even 100 times less.

[0150] In the case of embodiments in which the targeted substrate isdegraded to an inactive form by the adzyme, the potency may be expressedin terms of “HL₅₀”, e.g., the concentration of adzyme required to reducethe half-life (T½) in vivo of the targeted substrate by 50 percent. Themore potent and selective the adzyme is, the lower the HL₅₀concentration is relative to the catalytic domain alone. In certainpreferred embodiments, the HL₅₀ of the adzyme is at least 2 times lessthan the catalytic domain alone, and more preferably at least 5, 10 oreven 100 times less.

[0151] In certain embodiments, the adzyme has a catalytic efficiency forthe catalyzed reaction with the targeted substrate of at least 10⁴M⁻¹sec⁻¹, and even more preferably at least 10⁵ M⁻¹sec⁻¹ or even atleast 10⁶ M sec⁻¹.

[0152] In certain embodiments, the adzyme has a catalytic efficiency forthe catalyzed reaction with the targeted substrate at least 5 timesgreater than the catalytic domain alone, and even more preferably atleast 10, 50 or even 100 times greater.

[0153] In certain therapeutic applications, it will be important tobalance the potency and specificity of an adzyme. A good balance ofpotency and specificity can be achieved through the following designcriterion:

k _(cat) ^(ES) /K _(m) ^(E) =j _(iff) /as/[S] _(eff)

[0154] In adzyme embodiments designed with this criterion, the catalyticdomain will be very weak, in some cases having a catalytic efficiency aslow as 100, 10, or 1 M⁻¹s⁻¹, or even lower. Thus, adzymes designed tobalance potency and specificity should be derived from weak enzymedomains.

[0155] In certain embodiments, the k_(off) rate of the targeting moietywill be similar for the substrate and the adzyme reaction product, andit will be desirable to optimize the k_(off) rate for high substrateaffinity and rapid release of the product when bound to the address. Inthese embodiments, the optimal k_(off) rate may be 0.001 sec⁻¹, 0.01sec⁻¹, 0.1 sec⁻¹, or greater, and can be approximated by:$k_{{off},{optimal}}^{AS} \approx \sqrt{k_{on}^{AS}{{\frac{k_{cat}}{K_{m}^{E}}\lbrack S\rbrack}_{eff}\lbrack S\rbrack}}$

[0156] when [S]_(off)<<K_(m) ^(E), wherein K_(m) ^(E) is the enzyme'sK_(m) (not the adzyme's). The k_(on) ^(AS) (k_(on) of adzyme) above isthe same as k₁ in Equation 2 below.

[0157] For a fusion protein of two domains both of which independentlybind the substrate, the “effective concentration of a substrate,”[S]_(eff), is the quotient of the overall association equilibriumconstant for the fusion protein binding to its substrate and the productof the association equilibrium constants for the two, independentaddress domains binding to the substrate. This definition follows FIG. 1and Equation 2 in Zhou, J. Mol. Biol. (2003) 329, 1-8. Each of the threeequilibrium constants required to determine [S]_(eff) can be measuredvia standard binding assays. In performing kinetic analysis, it isfurther assumed that the microscopic off rates for each domain in afusion protein are not affected by the presence of the linker.

[0158] In certain embodiments, the adzyme has a K_(m) for catalyzedreaction with the targeted substrate at least 5 times less than thecatalytic domain alone, and even more preferably at least 10, 50 or even100 times less.

[0159] Broadly, the adzyme may be designed to interact with anybiomolecule target provided the site of enzymatic attack and the bindingsite for the address are solvent accessible. Thus, both the targetedbiomolecule and the binder for the address may be a soluble biomoleculeor a membrane-bound biomolecule. The target may be intracellular,although extracellular targets are more accessible to protein constructsand are therefore preferred.

[0160] Referring to FIG. 1, schematic diagrams illustrative of variousstructures which can exploit the invention are set forth as FIGS. 1Athrough 1K. In 1A, perhaps the simplest adzyme, an address (ADD) iscovalently linked to a catalytic domain (CAT). Such a construct may beembodied as two separate globular protein domains attached by a flexibleor rigid linker as illustrated, or by a single globular protein whereinone portion of the molecular surface functions as the address andanother as a catalytically active site. In FIG. 1B, the domains arecomplexed, i.e., each comprises a surface that reversibly binds to asurface on its partner. In FIGS. 1C through 1F, the address andcatalytic domains are associated via a chaperone protein, with either orboth linked to the chaperone via covalent bonds such as a linker ornoncovalent protein-protein complexation. In FIGS. 1G and 1H, each ofthe address and catalytic domains is linked, covalently or noncovalently, to a chaperone protein domain, and the chaperone domains arenoncovalently complexed together.

[0161]FIGS. 1I and 1J illustrate one way to exploit the recruitmentembodiment of the invention. These constructs, as illustrated, comprisesan address linked (covalently or non covalently) to a chaperone protein,which defines a binding surface specific for a predetermined catalyticdomain, i.e., an enzyme either already present in a body fluid or one coadministered with the construct. This type of construct functions byrecruiting the enzyme to the vicinity of the targeted biomolecule,mediated by the affinity of the address for the target so that the fullyfunctional adzyme is assembled in vivo. Of course, such enzymerecruiting constructs could also be embodied in other forms providedthey have a binding surface serving as an address that binds to thebinding site on or adjacent the target, and a binding surface thatserves to bind specifically to an enzyme. For example, a recruitmentconstruct may be embodied as a single globular protein, or as a globularprotein defining a binding surface for a catalytic domain and a smallmolecule with affinity for the target linked to it through a length ofbiocompatible polymer.

[0162] After the enzymatic reaction is complete, the adzymedisassociates from the target (now converted to a product) and moves onto bind to and act on another molecule of the target, creating turnover.As a result of this feature of the adzymes, the potency of the drugconstructs is not dependant directly on drug/target stoichiometry. Thisprovides a significant engineering advantage and can permit avoidance oftoxicity issues associated with the use of antibodies or small moleculedrugs inhibiting soluble biomolecules associated with a disease.

[0163] The equations below illustrates two possible adzyme (A-E)interactions between an address (A) and its binding site on a targetedbiomolecule (S), and between the adzyme's enzymatically active site (E)and the targeted substrate (S) to make product (P).

[0164] Reaction 1 is the normal catalytic reaction, where the address isnot involved, such as might occur with a substrate that does not displaya binding site for the address. In the presence of a local concentrationof both the adzyme (A-E) and the biomolecule (S) the targeted substratehas an on rate k₁ for the enzyme pocket (E), forms a complex A-E---Swith the pocket, and is converted at a rate dependent on k_(cat) toproduct P and released.

[0165] Reaction 2 occurs when the binding site on the targeted substrateS binds to the adzyme through formation of an address: binding siteinteraction (with an affinity that may be higher than the E---Saffinity), forming a complex S---AE with on rate k₁. Presuming asuitable structure of the adzyme, e.g., the length of the linker orstereochemistry of the complex and its target permits, this complex canenter an intermediate state at rate k₂ where the targeted substrateinteracts simultaneously with the address and the enzyme pocket. In thisstate the targeted substrate is converted to product P at a rategoverned by k_(cat), and then disassociates from the adzyme at rate k₃.

[0166] The functioning and structure of various forms of adzymes may beunderstood better with reference to FIGS. 2A-2J. FIG. 2A depicts anadzyme in situ at a moment when it has bound to its intendedbiomolecule. In this case the adzyme is embodied as a single globularprotein which defines a catalytic domain (CD) having an enzymaticallyactive site and an address (AD) defined by a separate surface on theprotein. The address binds reversibly with a binding site, in this caseembodied as a surface on the targeted biomolecule. The targetedsubstrate site is vulnerable to immediate enzymatic attack by theenzymatically active site of the catalytic domain.

[0167]FIG. 2B shows a construct similar to FIG. 2A except that theaddress is a small molecule attached to the catalytic domain by aflexible linker that binds reversibly directly with a binding site onthe intended targeted biomolecule.

[0168]FIG. 2C is an adzyme similar to 2B in which the address and thecatalytic domain are attached by a flexible leash. Binding of theaddress domain to the binding site, here again illustrated as a portionof the targeted biomolecule, serves effectively to increase the localconcentration of the catalytic domain in the region of the target, asillustrated. The address domain and the catalytic domain may be linkedvia a flexible linker, or a more rigid structure (not shown) such thatbinding of the address domain serves to pose the catalytic domain inposition to induce chemical change in its targeted biomolecule.

[0169] The adzyme of FIG. 2D is similar to FIG. 2C, except that thebinding site and the targeted biomolecule are separate molecularspecies, here illustrated as being lodged in a membrane, such as a cellmembrane. As in the embodiments of FIGS. 2A-2C, binding of the addressdomain to the recognition site of what here functions as a attractantmolecule serves to effectively increase the local concentration of thecatalytic domain in the region of the target. Where the concentration oftwo proteins on a cell is significant, especially in cases where theyare known to interact in lipid rafts or the like, one molecule can beused as the binding site to attract the construct to the other moleculethat will be catalytically modulated.

[0170] The adzyme of FIG. 2E is similar to FIG. 2C, except that theaddress domain and the catalytic domain are non-covalently associateddirectly to each other. Examples of this type of association includedimerization, optionally stabilized by disulfide linkages, hybridizationof complementary nucleotides, or protein-protein complexation of thetype that is ubiquitous within cells.

[0171]FIG. 2F shows an embodiment of an adzyme similar to FIG. 2E,except that the address domain is designed to bind to an attractantbiomolecule separate from but complexed to the targeted biomolecule.Nevertheless, binding increases the effective concentration of thetarget and its substrate site in the vicinity of the catalytic domain asshown.

[0172]FIG. 2G is the same as FIG. 2F except that the targetedbiomolecule is complexed with a separate protein displaying the bindingsite through a third, complexing protein.

[0173]FIG. 2H illustrates an embodiment of an adzyme in which theaddress and the catalytic domain are non-covalently associated through athird, chaperone protein, to form an active complex. Its intendedtargeted biomolecule is illustrated as being embedded in a lipidbilayer, and the binding site is illustrated as residing on a separatemolecule in the lipid bilayer, similar to FIG. 2D. Again, bindingnevertheless increases the effective concentration of the target and itssubstrate site in the vicinity of the catalytic domain.

[0174]FIG. 2I illustrates an embodiment of an adzyme similar to FIG. 2H,except that the address domain binds to a binding site directly on thetargeted biomolecule.

[0175]FIG. 2J is similar to FIG. 2G, except that the address domain andcatalytic domain of the adzyme are held together via complexation with achaperone protein. In all construct where the AD and CD are noncovalently complexed, the surface on the address domain that binds tothe catalytic domain (or a chaperone protein) may be the same ordifferent from the one that binds to the binding site on the target ortrigger molecule.

[0176] A further optional feature of adzymes is “engineeredcontingency,” that is, creation of a family of adzymes that becomecapable of reacting with their target in the presence of the target oranother triggering or attractant molecule having an affinity for theaddress. FIG. 1K illustrates the fundamental idea behind the contingentadzyme. As illustrated, the address has an affinity for the catalyticdomain and is configured so that it can bind to it and inhibit itsenzymatic activity. In the presence of the target, a competition for theaddress ensues, freeing the catalytic domain to induce chemical changein its intended target.

[0177] Stated differently, contingent adzyme constructs are inactive(have low enzymatic activity) in the absence of a triggering molecule,but become active in the presence of the triggering molecule, e.g., thetarget (see Legendre D. et al. (1999) Nature Biotechnology 17:67-72;Legendre D. et al. (2002) Protein Science 11:1506-1518; Soumillion P.and Fastrez J. (2001) Current Opinion in Biotechnology 12:387-394). Thistype of adzyme also requires a catalytic domain and an address. However,in this case, binding of the address has the effect of freeing up thecatalytic site of the catalytic domain to enhance its activity. This maybe achieved in several ways, illustrated by way of example in FIGS. 3Athrough 3G, which are described in more details in the contingent adzymesection.

[0178] In addition to the address and catalytic domains, and theoptional chaperone proteins, linkers and other structures defining therelationship of these parts, an adzyme may further comprise one or morefusion partners operatively linked to any of its components, e.g.,N-terminal or C-terminal fusions, or added or substituted sequences inloops on protein domains. Adzymes may also include polymeric sidechains, small molecules, or metal ions. These moieties may, for example,restrict the adzyme to a conformationally restricted or stable form;serve as a targeting sequence allowing the localization of the adzymeinto a sub-cellular or extracellular compartment; assist in thepurification or isolation of either the adzyme or the nucleic acidsencoding it; serve to confer a desired solubility on the adzyme; orconfer stability or protection from degradation to the adzyme or thenucleic acid molecule(s) encoding it (e.g., resistance to proteolyticdegradation). The adzyme may comprise one or any combination of theabove fusion partners as needed.

[0179] The fusion partners can, for example, be (histidine)₆-tag,glutathione S-transferase, protein A, dihydrofolate reductase, Tag•100epitope (EETARFQPGYRS; SEQ ID NO:1), c-myc epitope (EQKLISEEDL; SEQ IDNO:2), FLAG®-epitope (DYKDDDK; SEQ ID NO:3), lacZ, CMP(calmodulin-binding peptide), HA epitope (YPYDVPDYA; SEQ ID NO:4),protein C epitope (EDQVDPRLIDGK; SEQ ID NO:5) or VSV epitope(YTDIEMNRLGK; SEQ ID NO:6).

[0180] The fusion partner may also be a membrane translocation domain,i.e., a peptide capable of permeating the membrane of a cell and whichis used to transport attached peptides into or out of a cell in vivo.Membrane translocation domains that may be used include, but are notlimited to, the third helix of the antennapedia homeodomain protein andthe HIV-1 protein Tat or variants thereof. Additional membranetranslocation domains are known in the art and include those describedin, for example, Derossi et al., (1994) J. Biol. Chem. 269, 10444-10450;Lindgren et al., (2000) Trends Pharmacol. Sci. 21, 99103; Ho et al.,Cancer Research 61, 474-477 (2001); U.S. Pat. No. 5,888,762; U.S. Pat.No. 6,015,787; U.S. Pat. No. 5,846,743; U.S. Pat. No. 5,747,641; U.S.Pat. No. 5,804,604; and Published PCT applications WO 98/52614, WO00/29427 and WO 99/29721.

[0181] A. Exemplary Targeting Moieties

[0182] It will be appreciated that a wide range of entities can be usedas targeting moieties in the subject adzymes. Fundamentally, thetargeting moiety reversibly binds to a predetermined feature (“addresssite”) associated with the targeted substrate. The targeting moietypresents one or more surfaces having chemical characteristics (e.g.,hydrophobic, steric and/or ionic) which permit it to bind selectively,or relatively selectively, with the address site. In many embodiments,the address will be a modular protein (including peptide) domain whichis provided in association with the catalytic domain. For example, thetargeting moiety can be an antibody, or a fragment of an antibody whichretains the ability to bind to the address site. Accordingly, thetargeting moiety can be derived from such antibody and antibodyfragments as monoclonal antibodies, including Fab and F(ab)2 fragments,single chain antibodies (scFv), diabodies, and even fragments includingthe variable regions of an antibody heavy of light chain that binds tothe address site.

[0183] Other examples of proteins that can be suitably adapted for usein the subject adzymes including ligand binding domains of receptors,such as where the targeted substrate of the adzyme is the receptorligand. Conversely, the targeting moiety can be a receptor ligand wherethe adzyme is directed to the receptor as the targeted substrate. Suchligands include both polypeptide moieties and small molecule ligands.

[0184] In still other embodiments, the targeting moiety can be anengineered polypeptide sequence that was selected, e.g., syntheticallyevolved, based on its kinetics and selectivity for binding to theaddress site.

[0185] The targeting moiety can also be a polyanionic or polycatonicbinding agent, such as an oligonucleotide, a polysaccharide, a polyaminopeptide (such as poly-aspartate, poly-glutamate, poly-lysine orpoly-arginine). In certain embodiments, such targeting moieties maintaina number of either negative or positive charges over their structure atphysiological pH. The address may also be a protein nucleic acid (PNA),a lock nucleic acid (LNA) or a nucleotide sequence, such as a singlestrand of DNA or RNA.

[0186] The targeting moiety may also be a small molecule that has beenselected based on the kinetics and selectivity it displays for bindingto an address site associated with the targeted substrate.

[0187] There are a variety of well-known techniques for generatinglibraries of polypeptide/peptide, nucleic acid (aptamer) and smallmolecule moieties that can be used to identify molecules having theappropriate specificity, selectivity and binding kinetics for use in anyparticular adzyme. For example, such techniques as described in U.S.Pat. Nos. 6,258,558 titled “Method for selection of proteins usingRNA-protein fusions” and 5837500 titled “Directed evolution of novelbinding proteins” can be readily adapted for use in identifying peptideor polypeptide targeting moieties for use in generating the subjectadzymes. Likewise, the preparation of aptamers previously described inthe art can be adapted for generating appropriate targeting moieties.See, for example, Tuerk Science 249:505-510 (1990); Klug Mol BiolReports 20:97-107 (1994); and Morris et al, PNAS 95:2902-2907 (1998), aswell as U.S. Pat. Nos. 5,843,701 and 5,843,653.

[0188] The address may be at least about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 70, 80, 90 or 100 amino acid residues long. Ranges usinga combination of any of the foregoing recited values as upper and/orlower limits are intended to be included in the present invention.

[0189] In certain preferred embodiments, the dissociation constant(K_(d)) for binding to the address site is lower (higher affinity)and/or the K_(off) rate is slower when the address site is bound to theunmodified targeted substrate relative to when it is bound to the adzymereaction product (e.g, the targeted substrate that has been acted on bythe catalytic domain). That is, conversion of the targeted substrate toan adzyme reaction product reduces the affinity of the targeting moietyfor the address binding site and promotes dissociation of the adzymefrom the reaction product. In certain embodiments: the K_(d) of thetargeting moiety for the adzyme reaction product relative to thetargeted substrate is at least 5 times greater, and even more preferably10, 100 or even 1000 times greater; and/or the K_(off) rate of thetargeting moiety for the adzyme reaction product is at least 5 timesfaster, and even more preferably 10, 100 or even 1000 times fasterrelative to the K_(off) at rate for the targeted substrate.

[0190] In certain embodiments of direct adzymes, the address site andsubstrate site are overlapping in the sense that binding of thetargeting moiety to the targeted substrate interferes with the abilityof the catalytic domain to act on the targeted substrate site. Thisinterference may be the result of steric occlusion, or the lack offlexility in the adzyme and/or targeted substrate to permit bothportions of the adzyme to simultaneously interact with the targetedsubstrate. In other embodiments, the address and substrate sites arespaced sufficiently apart, and the adzyme has sufficient stericflexibility, that dissociation of the targeting moiety is not requiredfor the adzyme to modify the targeted substrate. In many embodiments,the adzyme will be designed such that there is functional cooperativitybetween the catalytic domain and targeting moiety, particularlyresulting from appropriate selection of linker(s) between the twocomponents, such that the affinity oft he resulting adzyme is at least 2times greater than the sum of the affinities of the catalytic domain andtargeting moiety, and even more preferably at least 5, 10, 100 or even500 times greater.

[0191] In some instances, the targeting moiety itself interferes withthe activity of the targeted substrate. For example, the targetingmoiety may be a blocking or neutralizing agent that inhibits anintrinsic activity or interaction mediated by the targeted substrate. Insuch cases, the adzyme with preferably be at least 5 times more potentan inhibitor, and even more preferably at least 10, 100 or even 1000times more potent than the targeting moiety alone.

[0192] In other embodiments, the targeting moiety does not itself haveany significant effect on the activity of the targeted substrate.

[0193] Where there are more than one possible substrate site of thecatalytic domain on a targeted substrate, such as more than onesubstrate recognition sequences for a proteolytic domain, the targetingmoiety can be selected to enhance the selectivity/preference of theadzyme for one of the sites. This can be accomplished, for example, byusing a targeting moiety that binds to the targeted substrate in amanner that sterically interferes with the catalytic domain's ability toact at one of the sites. In other embodiments, the targeting moiety canbe used to increase the concentration of the catalytic domain in theproximity of the desired substrate site.

[0194] In certain embodiments, the adzyme may include two or moreaddress/targeting moieties, which may be the same or different (i.e.,their respective K_(d) may be the same or different). In suchembodiments, the effective K_(d) of the adzyme for the targetedsubstrate may be as low as 10⁻¹⁵M (femtomolar), when the effectivesubstrate concentration [S]eff is greater than the highest individualK_(d) of the addresses (or targeting moieties).

[0195] In certain embodiments, the targeting moiety binds to a atargeted substrate which is soluble under the reaction conditions, suchas a soluble protein. In many cases, these soluble protein substrateswill be present in the reaction milieu at relatively low concentrations,such as less than 0.1 μM, and often at less than 10 nM. In suchembodiments, and certain others herein, it may be desirable to select atargeting moiety which, when provided in the adzyme, results in a directadzyme having a relative fast k_(on) for binding to the targetedsubstrate, e.g., a k_(on) of 10³ M⁻¹s⁻¹ or greater, e.g., at least 10⁴M⁻¹s⁻¹, 10⁵ M⁻¹s⁻¹ or even 10⁶ M⁻¹s⁻¹.

[0196] (i) Exemplary Targeted Biomolecules

[0197] In certain embodiments, the subject adzymes are directed tobiologically active molecules (“targeted biomolecule”), e.g., includingsolvent accessible extracellular and intracellular substrates, as wellas extracellular or cytoplasmic portions of membrane associatedsubstrates. These include, but are not limited to, substrates from amongsuch classes as protein and peptide substrates, nucleic acids, lipids,small molecules including extracellular factors (such as steroids andneurotransmitters) and intracellular second messengers (such asphosphorylated inositol and cAMP). By modifying the functionalperformance of a targeted substrate of biological relevance, the subjectadzymes can be used to alter such cellular processes as gene expression,morphology, cell adhesion, growth, proliferation, migration,differentiation and/or viability of cell.

[0198] Taregeted substrates can be modified by the adzyme so as toproduce one or more products having one or more differences inbiological activities relative to the targeted substrate (including, forexample, the elimination of all or near all biological activity of thetargeted substrate). For instance, for targeted substrates which arethemselves enzymes, the subject adzymes can be used to alter theintrinsic enzymatic activity of those targeted substrates. Toillustrate, an adzyme can used to inhibit such proteases as elastase (inthe treatment of cystic fibrosis, acute respiratory distress syndrome,and emphysema) or matrix metalloproteases involved in metastasis. Inother embodiments, the adzyme alters the ability of a targeted substrateto interact with other biological moieties, e.g., such as by alteringreceptor-ligand interactions, protein-protein interactions,protein-lipid interactions, protein-DNA or protein-RNA interactions toname but a few. In this respect, the adzyme can be used to increase ordecrease the intrinsic activity or binding activity of the targetedsubstrate. Adzymes can also be used to alter the half-life orbiodistribution of a targeted substrate.

[0199] In certain instances, the adzyme can be used to covert a targetedsubstrate into a functional antagonist of the unmodified biomolecule.Merely to illustrate, in the case of a polypeptide factor that actsthrough a receptor interaction, rather than generate a product that isunable to interact with the cognate receptor of the targeted substrate,the adzyme can be selected so as to alter the targeted substrate toproduce a product that retains the ability to bind to the receptor butnot induce the level of receptor activation possible by the unmodifiedtargeted substrate. In this way, the adzyme inhibits the function of thepolypeptide factor by (a) reducing the concentration of the polypeptidefactor, and (b) generating an antagonist which reduces the effectiveconcentration of receptor for the polypeptide factor. In preferredembodiments of this system, the product has a K_(i) of 10 μM or less forinhibiting an activity of the targeted substrate, and even morepreferably has a K_(i) less than 10 μM, 100 nM, 10 nM or even 1 nM.

[0200] (a) Extracellular Targets

[0201] In certain embodiments, the adzyme is directed to anextracellular target, including target molecules that are typicallylocated entirely outside of a cell and target molecules that areinserted into a cellular membrane but have a portion that is exposed tothe extracellular environment. Several categories of extracellulartargets are recognizable, including, for example, diffusibleextracellular molecules (e.g., growth factors, serum proteins,antibodies, any diffusible small molecule, extracellular nucleotides,lipids), extracellular molecules that are part of an insoluble aggregate(e.g., β-amyloid protein, constituents of atherosclerotic plaques,insoluble fibrin fibers), membrane associated proteins and othermembrane bound moieties (e.g., transmembrane proteins, lipids, membraneassociated polysaccharides), and constituents of or associated with anorganized extracellular matrix.

[0202] Accordingly, the subject adzymes can be used to alter, e.g.,inhibit or potentiate, such cell-surface mediated signaling as autocrinesignaling (self-signaling), paracrine signaling (between nearby cells),and/or endocrine signaling (over a long distance, usually via thebloodstream or other bodily fluid). The subject adzymes can also be usedto alter juxtacrine signaling, e.g., signaling consequences of cellcontact.

[0203] Various illustrative examples of different types of extracellulartargets are provided in Table I, below, along with associated conditionsthat antagonistic adzymes may be used to treat. TABLE I Examples ofvarious extracellular targets and associated conditions. TargetDisease/Condition TNF receptor Inflammation, arthritis, autoimmunethyroid disease, ischemic heart disease TNF-α and β Inflammation,arthritis, autoimmune thyroid disease, ischemic heart disease IL-2receptor Ischemic heart disease Aldosterone Cardiovascular heart diseaseAmyloid beta-peptide Alzheimer's disease [Abeta(1-42)] TransthyretinAlzheimer's disease Erythropoietin benign erythrocytosis ProstaglandinNeurodegeneration Cholesterol Heart disease Retinoid Xhepatogastroenterological diseases Apolipoprotein B-100 Coronary heartdisease Homocysteine Cardiovascular disease Insulin DiabetesApolipoprotein A1 Heart disease Apolipoprotein CII HyperlipidemiaApolipoprotein CII heart disease Apolipoprotein E Cardiovascular diseaseApolipoprotein E Alzheimer's disease CD4 Immune response CD4 receptorImmune response/HIV infection CCR5 Immune response/HIV infection SBR1HDL receptor/coronary heart disease Annexin V Clot formation, ApoptosisFibrin Wound healing, clot formation

[0204] Among the diffusible extracellular molecules, furthersubcategories are recognizable. In a preferred embodiment, a target ofan adzyme is an extracellular signaling molecule, meaning a moleculethat is produced by one cell with the primary effect of triggering aresponse in another cell. Examples of extracellular signaling moleculesinclude most growth factors and cytokines, neurotransmitters, hormones,and prostaglandins. Many extracellular signaling molecules are actuallypart of a larger assemblage that carries out the signaling function; forexample, TGF-β1 contains two 112 amino acid chains that are linked by adisulfide bond, and either of the two polypeptide chains may beconsidered to be extracellular signaling molecules that are targeted byan adzyme. Antibodies are explicitly not included in the term“extracellular signaling molecule”.

[0205] In certain embodiments, an extracellular signaling molecule is amolecule that binds to an extracellular portion of a membrane boundreceptor and triggers a signal transduction event in the cell. Incertain embodiments, an extracellular signaling molecule is a moleculethat enters a cell and binds to an intracellular receptor to trigger asignal transduction event in the cell (e.g., steroid hormones, harpinproteins of various bacterial pathogens).

[0206] In a particularly preferred embodiment, the target of an adzymeis an extracellular polypeptide signaling molecule, e.g., as may befound in biological fluid(s), such as a growth factor, cytokine,polypeptide hormone or the like. In certain preferred embodiments, thetargeted substrate is a signaling molecule, particularly a polypeptidesignaling molecule, present in serum or other bodily fluid at aconcentration of less than 1 μM, and even more preferably less than 0.1μM, 10 nM, 1 nM, 0.1 nM, 10 pM or even 1 pM. The catalytic domain ischosen so as to modify the signaling molecule in a manner that altersits interaction with a cognate receptor (e.g., abrograting binding orlimiting receptor activation), ability to form protein complexes withother soluble factors, half-life and/or biodistribution.

[0207] In certain preferred embodiments, the adzyme alters the level ofsignal transduction induced by an extracellular factor. The term “signaltransduction” is intended to encompass the processing of physical orchemical signals from the extracellular environment through the cellmembrane and into the cell, and may occur through one or more of severalmechanisms, such as activation/inactivation of enzymes (such asproteases, or enzymes which may alter phosphorylation patterns or otherpost-translational modifications), activation of ion channels orintracellular ion stores, effector enzyme activation via guaninenucleotide binding protein intermediates, second messenger generation(e.g., GTP hydrolysis, calcium mobilization, formation of inositolphosphates, cyclic nucleotides, sugar nucleosides or dissolved gasessuch as NO or O₃), redistribution of intracellular ions (Ca⁺², Zn⁺²,Na⁺, K⁺), and/or direct activation (or inhibition) of a transcriptionalfactor. Signal transduction may result in physiological changes to thecell, such as changes in morphology, cell adhesion, chemotaxis, drugresistance, growth, proliferation, death (apoptosis or necrosis),effector function, secretion of matrix, etc.

[0208] The induction of intracellular signals by the binding of anextracellular signaling molecule, such as a soluble growth factor, to amembrane-spanning receptor is of considerable biological importance. Inmany cases, promotion of receptor-receptor interactions by proteinfactors is a key initial step in the induction of a signal transductionprocess. In certain preferred embodiments, the subject adzymes can beused to alter the biological function/performance of an inductiveprotein factor, such as a protein factor selected from one of theprotein factor superfamilies known as (i) four-helix bundle factors,(ii) EGF-like factors, (iii) insulin-like factors, (iv) β-trefoilfactors and (v) cysteine knot factors. Exemplary substrates within inthese classes are listed in Table II. TABLE II Growth factor structuralsuperfamilies Family Subclass Examples Four-helix bundle Short chainIL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-13, IL-15, M-CSF, GM-CSF Longchain GH, LIF, G-CSF, IL-6, IL-12, EPO, OSM, CNTF Interferon IFNβ, IFNγEGF-like EGF, TGFα, heregulin Insulin-like Insulin, IGF1, IGF2 β-trefoilFGF, IL-1 Cysteine knot NGF, PDGF, TGFβ proteins

[0209] Examples of particular extracellular signaling molecules andconditions associated with these targets which may be treated using anappropriate adzyme are listed in Table III, below. TABLE III Examples ofExtracellular Signaling Molecules Target Disease/Condition IL-1α and βInflammation, Arthritis, inflammatory bowel disease IL-4 Asthma,allergic airway disease IL-5 Asthma, Allergic airway disease IL-6Inflammation, Kaposi's sarcoma IL-7 Immune response IL-8 Inflammatorydisease, Crohn's disease IL-18 Arthritis IL-9 Asthma IL-10 Colitis IL-11Crohn's disease, Ischemic heart disease TNF-α and β Inflammation,arthritis, autoimmune thyroid disease, ischemic heart disease VEGFCancer, Angiogenesis, Arthritis, Eales′ disease AldosteroneCardiovascular heart disease Somatostatin Grave's disease FibronectinUllrich′s disease Angiotensin Heart disease Erythropoietin Benignerythrocytosis Prostaglandins Neurodegeneration Interferon α and βImmune response Retinoid X hepatogastroenterological diseasesAdrenocorticotropic Cushing's disease Hormone Hepatocyte growth factorCardiovascular disease, periodontal disease Transforming growthGraft-versus-host disease, renal disease factor-beta 1 Transforminggrowth Coeliac Disease factor-beta Insulin-like growth factormacrovascular disease and hypertension in binding protein-1 type 2diabetes (IGFBP-1) VEGF-A Paget′s disease Platelet-derived Paget′sdisease endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) Insulin-like growth factor Inflammatory bowel disease I (IGF-I)IGF binding protein-3 Inflammatory bowel disease Insulin Diabetes EGFOncogenesis, Wound healing Vasoactive intestinal Inflammation peptide

[0210] In certain particularly preferred embodiments, the targetedsubstrate is an inflammatory cytokine, such as tumor necrosis factor(TNF-α), interleukin-6 (IL-6) or interleukin-1b (IL-1b), and the adzymecan be used therapeutically to reduce inflammation.

[0211] In certain other preferred embodiments, the targeted substrate isa polypeptide hormone, such as Adrenocorticotrophic Hormone, AmylinPeptide, Bombesin, Calcitonin, Cholecystokinin (CCK-8), Gastrin,Glicentin, GLP-1, GLP-2, PYY, NPY, GIP, Glucagon, Human ChorionicGonadotrophin (α), Human Chorionic Gonadotrophin (β), Human FollicleStimulating Hormone (β2), Human Growth Hormone, Insulin, LuteinisingHormone, Pancreatic Polypeptide, Parathyroid Hormone, PlacentalLactogen, Proinsulin, Prolactin, Secretogranin II, Somatostatin,Thyroglobulin, Thyroid Stimulating Hormone, Vasoactive IntestinalPolypeptide.

[0212] Other exemplary substrates for the subject adzymes includepolypeptide factors selected from the group consisting of:Granulocyte-colony stimulating factor (G-CSF), Myelomonocytic growthfactor, Interleukin-3, Interleukin-7, Leukemia inhibitory factor (LIF),Oncostatin M, Ciliary neurotrophic factor (CNTF), cholinergicdifferentiation factor (CDF), Interleukin-4, Interleukin-13,Interleukin-16, Interleukin-17, Interferon-alpha (IFN-αα),Interferon-beta (IFN-β), IFN-tau (IFN-τ), Interferon-omega (IFN-Ω),Interleukin-5, Granulocyte-macrophage colony-stimulating factor(GM-CSF), Macrophage colony-stimulating factor (M-CSF), Interleukin-10,Interleukin 1-alpha (IL-1-α), Interleukin 1-beta (IL1-β), Gonadotropin,Nerve Growth Factor (NGF), platelet factor 4 (PF-4), bTG, GRO, 9E3,HLA-A2, macrophage inflammatory protein 1 1 alpha (MIP-1α), macrophageinflammatory protein 1 beta (MIP-1β), Melanoma growth stimulatingactivity (MGSA), 4-IBB Ligand, ADF, Autocrine Motility Factors, B61,Betacellulin, Cardiotrophin-1, CD27 Ligand, CD30 Ligand, CD40 Ligand,CeK5 Receptor Ligand, EMAP-II, ENA-78, Eosinophil Cationic Protein,Epiregulin, Erythrocyte-derived Growth-Promoting Factor, Erythropoietin,Fas Ligand, Fibrosin, FIC, GDNF, Growth/Differentiation Factor-5,Interleukin-1 Receptor Antagonist, Interleukin-3, Interleukin-6,Interleukin-7, Interleukin-9, Interleukin-11, Interleukin-12,Interleukin-13, Interleukin-14, Interleukin-15, Lymphotactin, LT-beta,Lymphotoxin, MCP-2, MCP-3, Megapoietin, Melanoma-derived GrowthRegulatory Protein, Monocyte Chemoattractant Protein-1, MacrophageMigration Inhibitory Factor, Neu Differentiation Factor, Oncostatin M,OX40 Ligand, Placenta Growth Factor, PLF, Scatter Factor, Steel Factor,TCA 3, Thrombopoietin, Vascular Endothelial Cell Growth Factor, BoneMorphogenetic Proteins, Interleukin-1 Receptor Antagonist, MonocyteChemoattractant Protein-1, c-Kit ligand (stem cell factor), CXCchemokines, CC chemokines, lymphotactin, and C-X3-C chemokines(fractalkine/neurotactin).

[0213] In other embodiments, the adzyme is directed to a substrateassociated with a cell surface, such as for altering the activity of acell surface receptor, ion channel, transporter, adhesion molecule,lipid, or extracellular matrix molecule such as a polysaccharide orglycosaminoglycan.

[0214] In certain preferred embodiments, the targeted substrate is acell surface receptor protein or ion channel. For instance, the adzymecan be designed to modify a ligand-binding receptor protein in a mannerthat alters ligand binding kinetics and/or signal transduction activityof the receptor. Receptor proteins which can be substrates for thesubject adzymes include any receptor or channel which interacts with anextracellular molecule (i.e. hormone, growth factor, peptide, ion) tomodulate a signal in the cell. To illustrate, the targeted substrate ofthe adzyme can be a site on a serpentine receptor (such as G proteincoupled receptor), an enzyme-linked receptor (such as a receptortyrosine kinase, receptor serine/threonine kinase, receptor proteintyrosine phosphatase, receptor guanylyl cyclase, or receptor nitricoxide synthase), or an ion channel (including an ion-channel-linkedreceptor). Exemplary receptors which can be altered by an adzyme includecytokine receptors; multisubunit immune recognition receptors (MIRR),chemokine receptors; growth factor receptors, or chemoattracttractantpeptide receptors, neuropeptide receptors, light receptors,neurotransmitter receptors, and polypeptide hormone receptors, to namebut a few. Further examples of cell surface receptors are provided inTable IV, along with associated conditions that may be treated byadministration of an appropriately targeted adzyme. TABLE IV Examples ofCell Surface Receptors Target Disease/Condition IL-1 receptorInflammation, Arthritis, inflammatory bowel disease TNF receptorInflammation, arthritis, autoimmune thyroid disease, ischemic heartdisease IL-2 receptor Ischemic heart disease EGF receptor CancerVascular endothelial Arthritis growth factor receptor VEGF receptorCancer Aldosterone receptor Cardiovascular heart disease Somatostatinreceptor Grave's disease Fibronectin receptor Ullrich's diseaseAngiotensin receptor Heart disease SBR1 HDL receptor/coronary heartdisease

[0215] Additional examples of cell surface associated or extracellularmatrix targets for the subject adzymes include cellular adhesionmolecules, such as selectins, integrins and other hemidesmosomalproteins, cadherins, laminins, CD44 isoforms, proteoglycans (such assyndecans), Ig superfamily (IgCAM) proteins, catenins (such as α, β, andγ catenins) and cadherins (such as E-cadherin or P-cadherin), galectins,collagens, elastins, fibrins, and the like.

[0216] In certain embodiments, the adzyme acts on a Cluster ofDifferentiation (CD) protein, such as CD1a, CD2 (LFA-2), CD3, CD4, CD5,CD6, CD7, CD8, CD9 (Motility-Related Protein-1), CD10 (CALLA), CD11b(Mac-1), CD11b, CD13, CD14, CD15, CD16, CD18 (b2), CD19, CD20, CD21,CD22 (BL-CAM), CD23, CD25 (Interleukin-2 Receptor), CD27, CD29 (b1),CD30, CD31 (PECAM-1), CD34 (Endothelial Cell Marker), CD35, CD37, CD38,CD39, CD40, CD40L (CD154), CD41 (GPIIb/IIIa), CD42b (GPIb), CD43, CD44(H-CAM), CD44 Variant 3, CD44 Variant 4, CD44 Variant 5, CD44 Variant 6,CD45 (Leucocyte Common Antigen), CD45RA, CD45RB, CD45RO, CD48, CD49b(VLA-2), CD49c (VLA-3), CD49f (VLA-6), CD50 (ICAM-3), CD51, CD54(ICAM-1), CD56 (NCAM), CD57, CD58 (LFA-3), CD61 (GPIIIa), CD61 (GPIIIa),CD62E (E-selectin), CD62L (L-selectin), CD62P (P-selectin), CD63(Melanoma Marker), CD66a (CEACAM1), CD66e (Carcinoembryonic Antigen),CD68, CD69, CD71 (Transferrin Receptor), CD72, CD74, CDw75, CD79a, CD81,CD82, CD83, CD95 (Fas), CD99 (MIC2), CD104, CD105 (Endoglin), CD106(VCAM-1), CD117 (c-kit Oncoprotein), CD134 (OX40), CD137, CD138(Syndecan-1), CD141 (Thrombomodulin), CD141 (Thrombomodulin), CD143(ACE), CD146 (MCAM), CD147 (EMMPRIN), CDw150 (SLAM), CD151 (PETA-3),CD154 (CD40L), CD162, CD163, CD166 (ALCAM), CD168 (RHAMM), or CD179a.

[0217] In certain preferred embodiments, the adzyme substrate is aselectin, e.g., a CD62 family protein. In other preferred embodiments,the adzyme substrate is an immunoglobulin superfamily protein (IgCAM),such as a CD2 family protein, CD22, CD31, CD48, CD50, CD54, CD56, CD58,CD66a, CD83, CD106, CD146, CD147, CDw150 or CD166. In still otherpreferred embodiments, the adzyme substrate is an integrin, such as CD49family, CD51, CD29, CD11b, CD18, CD41, CD61 or CD104.

[0218] Certain of the subject adzymes can be used to alter the activityof scavenger receptor class A (SR-A, CD204), scavenger receptor-BI(SR-BI) or CD36, which are cell surface proteins that mediate celladhesion to, and endocytosis of, various native and pathologicallymodified substances, and participate in intracellular signaling, lipidmetabolism, and host defense against bacterial pathogens.

[0219] Collagenolytic adzymes can be prepared, e.g., using collagenasecatalytic domains from hydrobionts, polycollagenase-K or Fermenkol, tocause deep hydrolysis of polypeptide substrates (native or partiallydenatured collagen types, elastin, fibrin, hemoglobin, and casein). Suchadzymes have use in both medical and cosmetological applications.

[0220] In certain embodiments, a ligand (or binding portion thereof) ofa receptor or other cell surface molecule may be employed as an addressmoiety. In certain embodiments, the adzyme can be associated with one ormore ligands effective to bind to specific cell surface proteins ormatrix on the target cell, thereby facilitating sequestration of theadzyme to target cells. For instance, the adzyme can be a fusion proteinthat also includes the ligand. Merely to illustrate, examples of ligandssuitable for use in targeting the adzymes of the present invention tospecific cell types are listed in the Table V below. TABLE V AdzymesSpecific for Various Cell Types Ligand Receptor Cell type folate folatereceptor epithelial carcinomas, bone marrow stem cells water solublevitamin receptor various cells vitamins pyridoxyl phosphate CD4 CD4 +lymphocytes apolipoproteins LDL liver hepatocytes, vascular endothelialcells insulin insulin receptor transferrin transferrin receptorendothelial cells galactose asialoglycoprotein liver hepatocytesreceptor sialyl-Lewis_(x) E, P selectin activated endothelial cellsMac-1 L selectin neutrophils, leukocytes VEGF Flk-1, 2 tumor epithelialcells basic FGF FGF receptor tumor epithelial cells EGF EGF receptorepithelial cells VCAM-1 a₄b₁ integrin vascular endothelial cells ICAM-1a_(L)b₂ integrin vascular endothelial cells PECAM-1/CD31 a_(v)b₃integrin vascular endothelial cells, activated platelets osteopontina_(v)b₁ integrin endothelial cells and a_(v)b₅ integrin smooth musclecells in atherosclerotic plaques RGD sequences a_(v)b₃ integrin tumorendothelial cells, vascular smooth muscle cells HIV GP 120/41 CD4 CD4⁺lymphocytes or GP120

[0221] In certain embodiments of adzymes intended to be antagonists of areceptor ligand, the adzyme will alter the receptor in a manner thatreduces the level of ligand-induced signal transduction, but will notsubstantially impair the ability of the receptor to bind to its cognateligand. In this manner, the adzyme antagonizes the ligand not only as aconsequence to the generation of loss-of-function receptors with regardto signal transduction, but also because the otherwise inactivatedreceptor can act as a competitive binding agent for sequestering theligand from still functional receptors. Alternatively, the adzyme can beselected to generate a receptor product which is constitutively active,e.g., in which case the adzyme acts may as an agonist of the receptor'sinductive ligand.

[0222] In certain embodiments, the intended substrate of the adzyme willbe a heteromeric receptor complex, e.g., receptor complexes involvingtwo or more different receptor subunits. For instance, receptors formost interleukins and cytokines that regulate immune and hematopoieticsystems belong to the class I cytokine receptor family. These moleculesform multichain receptor complexes in order to exhibit high-affinitybinding to, and mediate biological functions of, their respectivecytokines. In most cases, these functional receptor complexes sharecommon signal transducing receptor components that are also in the classI cytokine receptor family, such as the gp130 protein. Adzymes which arespecifically reactive with the unique receptor subunit(s), but which donot substantially impair the function of the common subunit, can be usedto enhance the selectivity of the adzyme as an antagonist of aparticular ligand.

[0223] Alternatively, adzymes that selectively inactivate the uniquereceptor subunits of other ligand-receptor complexes, e.g., those thatcompete with the formation of receptor complexes for the ligand ofinterest, can be agonists of ligand of interest.

[0224] In still other embodiments, an adzyme is targeted to anextracellular molecule that is part of a biomolecular accretion. Abiomolecular accretion is any undesirable assemblage of biomolecules,usually one that brings together components that are not typically foundin an assemblage together usually one that has grown over time by thesuccessive addition of material. Accretions are generally large enoughas to be non-diffusible (although clots are accretions that may diffusein the circulatory system) and are generally larger than the size of atypical host cell. Biomolecular accretions will often contain dead andliving cells as well as extracellular matrix. Examples of biomolecularaccretions include amyloid deposits, e.g., a β-amyloid peptide depositcharacteristic of Alzheimer's disease or a type II diabetes amyloiddeposit, a collagen deposit, a protein deposit, an atheroscleroticplaque, an undesirable fat mass, an undesirable bone mass, a blood clot,or a cyst. In certain embodiments, an adzyme is designed to target oneor more extracellular molecules of a biomolecular accretion and act onsuch targets in such a way as to cause the partial or completedissolution of the accretion. Examples of proteins that are oftenpresent in the amyloid deposits associated with Alzheimer's diseaseinclude amyloid β-peptide [Aβ(1-42)] and transthyretin. Proteinaggregation has been linked to several human diseases, includingAlzheimer's disease, Parkinson's disease, and systemic amyloidosis. Mostof these diseases are associated with the formation of highly orderedand beta-sheet-rich aggregates referred to as amyloid fibrils. Fibrilformation by WT transthyretin (TTR) or TTR variants has been linked tosystemic amyloidosis and familial amyloid polyneuropathy, respectively.Amyloid fibril formation by α-synuclein (α-syn) has been linked toneurodegeneration in Parkinson's disease. Atherosclerotic plaque maycontain a variety of different components. Examples of certaincomponents include: calcified substances (e.g., hydroxyapatite),cholesterol crystals, collagen matrix, macrophage foam cells, smoothmuscle cells, lipid-rich atheromatous material (particularly rich incholesterol and esters thereof), mast cells, matrix metalloproteinases(e.g., MMP-1 collagenase, MMP-2 and -9 gelatinases). Given thatatherosclerotic plaque rupture is associated with dangerous thromboticevents, it may be desirable to design an adzyme that stabilizes plaques(e.g. by targeting metalloproteinases in the plaque) or to employ aplaque-dissolving adzyme in combination with an anti-thrombotic agent,such as heparin.

[0225] Often, a biomolecular accretion combines various biomoleculesthat have appropriate roles in other parts of an organism; accordingly,it may be desirable to selectively target molecules that are primarilypresent in the accretion or to provide an adzyme with multiple differentaddress moieties that enhance adzyme concentration in the vicinity ofthe accretion.

[0226] (b) Intracellular Targets

[0227] In certain embodiments, an adzyme may be directed against anintracellular target. Examples of intracellular targets includeintracellular receptors (e.g., many steroid hormone receptors), enzymesthat are overexpressed or otherwise participate in an undesirablecondition, intracellular signaling proteins that participate in anundesirable condition (e.g., oncoproteins, pro-inflammatory proteins)and transcription factors.

[0228] In an exemplary embodiment, the adzyme alters a nuclear receptor.Many nuclear receptors may be viewed as ligand-dependent transcriptionfactors. These receptors provide a direct link between extracellularsignals, mainly hormones, and transcriptional responses. Theirtranscriptional activation function is regulated by endogenous smallmolecules, such as steroid hormones, vitamin D, ecdysone, retinoic acidsand thyroid hormones, which pass readily through the plasma membrane andbind their receptors inside the cell. The subject adzymes can be used,for example, to alter the responsiveness of a cell to a particularhormone or other nuclear receptor ligand, such as by degrading receptorcomplexes to inhibit response to a hormone of interest, or degradingsubunits for other receptor dimers that otherwise compete with theformation of receptor complexes for the hormone of interest (such thatthe adzyme is an agonist of that hormone).

[0229] Examples of certain intracellular targets are provided in TableVI, along with associated conditions that may be treated with anappropriately targeted adzyme. TABLE VI Examples of IntracellularTargets Target Disease/Condition aldosterone receptor Cardiovascularheart disease Erythropoietin benign erythrocytosis PPAR_(γ)hepatogastroenterological diseases Adrenocorticotropic Cushing's diseaseHormone Huntingtin protein Huntington's disease estrogen receptorCoronary heart disease, Liver disease glucose-6-phosphatase Glycogenstorage disease type 1 erythrocyte antioxidant Behcet's disease enzymeandrogen receptor Paget's disease platelet-derived Paget's diseaseendothelial cell growth factor/thymidine phosphorylase (PD- ECGF/TP) RbCancer P16 Cancer P21 Cancer P53 Cancer HIF-1 Cancer NF-κB Inflammatorydisease NF-κB Cell Death IκB Immune response

[0230] In embodiments involving an intracellular target, it willgenerally be desirable to have an adzyme that is produced within cellsor designed for entry into cells. In certain embodiments, the adzyme mayinclude one or more functionalities that promote uptake by target cells,e.g., promote the initial step of uptake from the extracellularenvironment. In one embodiment, a subject adzyme includes an“internalizing peptide” which drives the translocation of the adzymeacross a cell membrane in order to facilitate intracellularlocalization. The internalizing peptide, by itself, is capable ofcrossing a cellular membrane by, e.g., transcytosis, at a relativelyhigh rate. The internalizing peptide is conjugated, e.g., to an adzyme.In certain embodiments, the adzyme may be expressed from a nucleic acidthat is introduced into a cell, such as a viral vector or naked orencapsulated nucleic acid vector. Nucleic acids for the intracellularproductions of adzymes are described in the section entitled “Nucleicacid compositions”, below.

[0231] In one embodiment, an internalizing peptide is derived from theDrosophila antepennepedia protein, or homologs thereof. The 60 aminoacid long homeodomain of the homeo-protein antepennepedia has beendemonstrated to translocate through biological membranes and canfacilitate the translocation of heterologous peptides and organiccompounds to which it is couples. See for example Derossi et al. (1994)J Biol Chem 269:10444-10450; and Perez et al. (1992) J Cell Sci102:717-722. Recently, it has been demonstrated that fragments as smallas 16 amino acids long of this protein are sufficient to driveinternalization. See Derossi et al. (1996) J Biol Chem 271:18188-18193.The present invention contemplates an adzyme including at least aportion of the antepennepedia protein (or homolog thereof) sufficient toincrease the transmembrane transport.

[0232] Another example of an internalizing peptide is the HIVtransactivator (TAT) protein. This protein appears to be divided intofour domains (Kuppuswamy et al. (1989) Nucl. Acids Res. 17:3551-3561).Purified TAT protein is taken up by cells in tissue culture (Frankel andPabo, (1989) Cell 55:1189-1193), and peptides, such as the fragmentcorresponding to residues 37-62 of TAT, are rapidly taken up by cell invitro (Green and Loewenstein, (1989) Cell 55:1179-1188). The highlybasic region mediates internalization and targeting of the internalizingmoiety to the nucleus (Ruben et al., (1989) J. Virol. 63:1-8). Peptidesor analogs that include a sequence present in the highly basic region,such as CFITKALGISYGRKKRRQRRRPPQGS (SEQ ID NO:), can be used in theadzyme to aid in internalization.

[0233] Another exemplary adzyme can be generated to include a sufficientportion of mastoparan (T. Higashijima et al., (1990) J. Biol. Chem.265:14176) to increase the transmembrane transport of the adzyme.

[0234] While not wishing to be bound by any particular theory, it isnoted that hydrophilic polypeptides and organic molecules may be also bephysiologically transported across the membrane barriers by coupling orconjugating the polypeptide to a transportable peptide which is capableof crossing the membrane by receptor-mediated transcytosis. Suitableinternalizing peptides of this type can be generated using all or aportion of, e.g., a histone, insulin, transferrin, basic albumin,prolactin and insulin-like growth factor I (IGF-I), insulin-like growthfactor II (IGF-II) or other growth factors. For instance, it has beenfound that an insulin fragment, showing affinity for the insulinreceptor on capillary cells, and being less effective than insulin inblood sugar reduction, is capable of transmembrane transport byreceptor-mediated transcytosis and can therefore serve as aninternalizing peptide for the subject adzyme. Preferred growthfactor-derived internalizing peptides include EGF (epidermal growthfactor)-derived peptides, such as CMHIESLDSYTC (SEQ ID NO:) andCMYIEALDKYAC (SEQ ID NO:); TGF-beta (transforming growth factorbeta)-derived peptides; peptides derived from PDGF (platelet-derivedgrowth factor) or PDGF-2; peptides derived from IGF-I (insulin-likegrowth factor) or IGF-II; and FGF (fibroblast growth factor)-derivedpeptides.

[0235] Another class of translocating/internalizing peptides exhibitspH-dependent membrane binding. For an internalizing peptide that assumesa helical conformation at an acidic pH, the internalizing peptideacquires the property of amphiphilicity, e.g., it has both hydrophobicand hydrophilic interfaces. More specifically, within a pH range ofapproximately 5.0-5.5, an internalizing peptide forms an alpha-helical,amphiphilic structure that facilitates insertion of the moiety into atarget membrane. An alpha-helixinducing acidic pH environment may befound, for example, in the low pH environment present within cellularendosomes. Such internalizing peptides can be used to facilitatetransport of the subject adzyme, taken up by an endocytic mechanism,from endosomal compartments to the cytoplasm.

[0236] A preferred pH-dependent membrane-binding internalizing peptideincludes a high percentage of helix-forming residues, such as glutamate,methionine, alanine and leucine. In addition, a preferred internalizingpeptide sequence includes ionizable residues having pKa's within therange of pH 5-7, so that a sufficient uncharged membrane-binding domainwill be present within the peptide at pH 5 to allow insertion into thetarget cell membrane.

[0237] A particularly preferred pH-dependent membrane-bindinginternalizing peptide in this regard isXaa1-Xaa2-Xaa3-EAALA(EALA)4-EALEALAA-amide (SEQ ID NO:), whichrepresents a modification of the peptide sequence of Subbarao et al.(Biochemistry 26:2964, 1987). Within this peptide sequence, the firstamino acid residue (Xaa1) is preferably a unique residue, such ascysteine or lysine, that facilitates chemical conjugation of theinternalizing peptide to a targeting protein conjugate. Amino acidresidues Xaa2-Xaa3 may be selected to modulate the affinity of theinternalizing peptide for different membranes. For instance, if bothresidues 2 and 3 are lys or arg, the internalizing peptide will have thecapacity to bind to membranes or patches of lipids having a negativesurface charge. If residues 2-3 are neutral amino acids, theinternalizing peptide will insert into neutral membranes.

[0238] Yet other preferred internalizing peptides include peptides ofapo-lipoprotein A-1 and B; peptide toxins, such as melittin,bombolittin, delta hemolysin and the pardaxins; antibiotic peptides,such as alamethicin; peptide hormones, such as calcitonin,corticotrophin releasing factor, beta endorphin, glucagon, parathyroidhormone, pancreatic polypeptide; and peptides corresponding to signalsequences of numerous secreted proteins. In addition, exemplaryinternalizing peptides may be modified through attachment ofsubstituents that enhance the alpha-helical character of theinternalizing peptide at acidic pH.

[0239] Pore-forming proteins or peptides may also serve as internalizingpeptides herein. Pore forming proteins or peptides may be obtained orderived from, for example, C9 complement protein, cytolytic T-cellmolecules or NK-cell molecules. These moieties are capable of formingring-like structures in membranes, thereby allowing transport ofattached adzyme through the membrane and into the cell interior.

[0240] (c) Infective or Foreign Targets

[0241] An additional category of targets for an adzyme are targets thatare associated with an infective or otherwise undesirable foreign agent,such as protists, yeasts, bacteria, viruses and prions and variouscomplexes. In certain embodiments, an adzyme is targeted against avirulence factor that is exposed on the surface of a bacterium, such asa pilin or other adhesive protein, a flagellin, or other motilityprotein, a protein that facilitates bacterial cell entry into the hostcell cytoplasm. In certain embodiments, an adzyme is targeted so as todisrupt a structural component of a bacterial cell wall or membrane,sufficient to cause cell lysis. In certain embodiments, an adzyme istargeted against a protein or other component of a virus that isrequired for viral particle viability or entry into a host cell, e.g., aprotein of a viral coat or envelope. In another example, an adzyme maybe targeted against a toxin, a venom, an undesirable foreign chemical ora heavy metal.

[0242] (d) Molecules Targeted by Developed Therapeutic Agents

[0243] One novel approach to designing effective adzymes is to identifymolecules that are targeted by therapeutically active agents that act bybinding to the targeted molecules, such as monoclonal antibodies andsoluble receptor portions. In a preferred embodiment, the targetmolecule is a target for a FDA-approved, commercially availabletherapeutic binding agent. It is expected that a molecule which can beeffectively targeted by a binding agent may also be targeted by anadzyme that provides increased effectiveness relative to the bindingagent.

[0244] In certain embodiments, the adzyme is an antagonist of CD52. Suchadzymes can be used as part of a treatment for B cell chroniclymphocytic lymphoma (CLL). CD52 is a 21-28 kD glycoprotein expressed onthe surface of normal and malignant B and T lymphocytes, NK cells,monocytes, macrophages, and tissues of the male reproductive system.Campath® (Alemtuzumab) is a recombinant DNA-derived humanized CD52monoclonal antibody (Campath-1H). One problem associated with the use ofCampath is hematologic toxicity, which tend to occur when single dosesof greater than 30 mg or cumulative doses greater than 90 mg per weekare administered. Thus the subject adzyme, which can be administered ata much lower dose because of its catalytic nature, is expected to be abetter therapeutic alternative. The adzyme address domain may use thesame monoclonal antibody or functional derivative thereof (such as ascFv derivative as in the instant application), as is generally the casein adzyme treatment of other diseases described below. A panel ofproteases that are capable of efficiently digesting CD52 may be used asthe catalytic domain.

[0245] In certain embodiments, the adzyme is an antagonist of TNF-alpha.Such adzymes can be used as part of a treatment for Rheumatoidarthritis, inflammatory bowel disease (IBD), including Crohn's diseaseand and ulcerative colitis. Human TNF-alpha is a non-glycosylatedprotein of 17 kDa, while murine TNF-alpha is N-glycosylated. TNF-alphashows a wide spectrum of biological activities, and is found to be theimportant part of the whole IBD problem. Enbrel (etanercept; Immunex)and Remicade (infliximab; Centocor) are TNF-alpha antibodies that areused for severe cases of Rheumatoid arthritis and Crohn disease. The twodrugs are very similar in mechanism, as is Humira (adalimumab; Abbott),a very recently approved TNF antibody which is much more faithful tohuman antibody structure. The subject adzyme, which can be administeredat a much lower dose because of its catalytic nature, is expected to bea better therapeutic alternative. The adzyme address domain may use thesame monoclonal antibody or functional derivative thereof (such as ascFv derivative as in the instant application). A panel of proteasesthat are capable of efficiently digesting TNF-alpha may be used as thecatalytic domain.

[0246] In certain embodiments, the adzyme is an antagonist of theHER2/neu receptor. Such adzymes can be used as part of a treatment formetastatic breast cancer and/or recurrent or refractory ovarian orprimary peritoneal carcinoma with overexpression of HER2. The HER2 (orc-erbB2) proto-oncogene encodes a transmembrane receptor protein of 185kDa, which is structurally related to the epidermal growth factorreceptor 1 (EGFR1). HER2 protein overexpression is observed in 25%-30%of primary breast cancers. HERCEPTIN (Trastuzumab) is a recombinantDNA-derived humanized monoclonal antibody that selectively binds withhigh affinity in a cell-based assay (K_(d)=5 nM) to the extracellulardomain of HER2. The antibody is a humanized murine IgG1 kappa. Oneproblem associated with the use of HERCEPTIN administration is severehypersensitivity reactions (including anaphylaxis), infusion reactions,and pulmonary events. Thus the subject adzyme, which can be administeredat a much lower dose because of its catalytic nature, is expected to bea better therapeutic alternative. The adzyme address domain may use thesame monoclonal antibody or functional derivative thereof (such as ascFv derivative as in the instant application). A panel of proteasesthat are capable of efficiently digesting HER2 may be used as thecatalytic domain.

[0247] In certain embodiments, the adzyme is an antagonist of CD33. Suchadzymes can be used as part of a treatment for Acute myeloid leukemia(AML), the most common type of acute leukemia in adults. CD33 antigen isa sialic acid-dependent adhesion protein found on the surface ofleukemic blasts and immature normal cells of myelomonocytic lineage, butnot on normal hematopoietic stem cells. “Mylotarg” (gemtuzumabozogamicin for Injection) is a chemotherapy agent composed of arecombinant humanized IgG4, kappa antibody conjugated with a cytotoxicantitumor antibiotic, calicheamicin, isolated from fermentation of abacterium, Micromonospora echinospora ssp. calichensis. The antibodyportion of Mylotarg binds specifically to the CD33 antigen. Side effectsassociated with the use of Mylotarg includes hypersensitivity reactions,including anaphylaxis, infusion reactions, pulmonary events, andhepatotoxicity. Thus the subject adzyme, which can be administered at amuch lower dose because of its catalytic nature, is expected to be abetter therapeutic alternative. The adzyme address domain may use thesame monoclonal antibody or functional derivative thereof (such as ascFv derivative as in the instant application). A panel of proteasesthat are capable of efficiently digesting CD33 may be used as thecatalytic domain.

[0248] In certain embodiments, the adzyme is an antagonist of CD3. Suchadzymes can be used as part of a treatment for transplant rejection,such as acute renal, steroid-resistant cardiac, or steroid-resistanthepatic allograft rejection. OKT3 (or “muromonab-CD3”) is a murinemonoclonal antibody to the CD3 antigen of human T cells which functionsas an immunosuppressant. The antibody is a biochemically purified IgG2aimmunoglobulin. It is directed to the CD3 glycoprotein in the human Tcell surface which is essential for T cell functions. Modulated cells,which reversibly lose the expression of the CD3 T cell receptormolecular complex but still share the CD4 and CD8 antigens, have beenshown to be functionally immunoincompetent. Thus the subject adzyme,which can be administered at a much lower dose because of its catalyticnature, is expected to be a better therapeutic alternative. The adzymeaddress domain may use the same monoclonal antibody or functionalderivative thereof (such as a scFv derivative as in the instantapplication). A panel of proteases that are capable of efficientlydigesting CD3 may be used as the catalytic domain.

[0249] In certain embodiments, the adzyme is an antagonist ofgpIIb/IIIa. Such adzymes can be used as part of a treatment for Acutemyocardial infarction/unstable angina. Abciximab (ReoPro®), is the Fabfragment of the chimeric human-murine monoclonal antibody 7E3. Abciximabbinds to the glycoprotein (GP) IIb/IIIa (a_(IIb) ₃) receptor of humanplatelets and inhibits platelet aggregation. Abciximab also binds to thevitronectin (a_(v)b₃) receptor found on platelets and vessel wallendothelial and smooth muscle cells. The subject adzyme, which can beadministered at a much lower dose because of its catalytic nature, isexpected to be a better therapeutic alternative. The adzyme addressdomain may use the same monoclonal antibody or functional derivativethereof (such as a Fab or scFv derivative as in the instantapplication). A panel of proteases that are capable of efficientlydigesting gpIIb/IIIa may be used as the catalytic domain.

[0250] In certain embodiments, the adzyme is an antagonist of CD20. Suchadzymes can be used as part of a treatment for Non-Hodgkin's lymphoma(NHL), such as CD20 positive, follicular, Non-Hodgkin's lymphoma. TheCD20 antigen is found on the surface of normal and malignant Blymphocytes. The RITUXAN® (Rituximab) antibody is a geneticallyengineered chimeric murine/human monoclonal antibody directed againstthe CD20 antigen found on the surface of normal and malignant Blymphocytes. The antibody is an IgG1 kappa immunoglobulin containingmurine light- and heavy-chain variable region sequences and humanconstant region sequences. Rituximab has a binding affinity for the CD20antigen of approximately 8.0 nM. A second approved drug, ZEVALIN(Ibritumomab Tiuxetan), is the immunoconjugate resulting from a stablethiourea covalent bond between the monoclonal antibody Ibritumomab andthe linker-chelator tiuxetan[N-[2-bis(carboxymethyl)amino]-3-(p-isothiocyanatophenyl)-propyl]-[N-[2bis(carboxymethyl)amino]-2-(methyl)-ethyl]glycine.This linker-chelator provides a high affinity, conformationallyrestricted chelation site for Indium-111 or Yttrium-90. The antibodymoiety of ZEVALIN is Ibritumomab, a murine IgG1 kappa monoclonalantibody directed against the CD20 antigen. A third drug, Bexxar(tositumomab and iodine-131 tositumomab), is another approved drug forthe treatment of patients with CD20 positive, follicular, Non-Hodgkin'slymphoma, with and without transformation, whose disease is refractoryto Rituxan and has relapsed following chemotherapy. The subject adzyme,which can be administered at a much lower dose because of its catalyticnature, is expected to be a better therapeutic alternative. The adzymeaddress domain may use the same monoclonal antibodies or functionalderivative thereof (such as a Fab or scFv derivative as in the instantapplication). A panel of proteases that are capable of efficientlydigesting CD20 may be used as the catalytic domain.

[0251] In certain embodiments, the adzyme is an antagonist of RSV FProtein. Such adzymes can be used as part of a treatment for RSVinfection. SYNAGIS® (PALIVIZUMAB) is a humanized monoclonal antibody(IgG1k) produced by recombinant DNA technology, directed to an epitopein the A antigenic site of the F protein of respiratory syncytial virus(RSV). Palivizumab is a composite of human (95%) and murine (5%)antibody sequences. The subject adzyme, which can be administered at amuch lower dose because of its catalytic nature, is expected to be abetter therapeutic alternative. The adzyme address domain may use thesame monoclonal antibody or functional derivative thereof (such as a Fabor scFv derivative as in the instant application). A panel of proteasesthat are capable of efficiently digesting RSV F protein may be used asthe catalytic domain.

[0252] In certain embodiments, the adzyme is an antagonist of CD25. Suchadzymes can be used as part of a treatment for transplant rejection.Zenapax® (daclizumab) is an immunosuppressive, humanized IgG1 monoclonalantibody produced by recombinant DNA technology that binds specificallyto the alpha subunit)_(p55) 1 alpha, CD25, or T ac subunit) of the humanhigh affinity IL-2 receptor that is expressed on the surface ofactivated (but not resting) lymphocytes. The drug binds to the highaffinity IL-2 receptor, thus inhibiting the binding of Tac by IL-2, andthe activation of lymphocytes. Therefore, the monoclonal antibody actsas a pure binder inhibitor. The subject adzyme, which can beadministered at a much lower dose because of its catalytic nature, isexpected to be a better therapeutic alternative. The adzyme addressdomain may use the same monoclonal antibody or functional derivativethereof (such as a Fab or scFv derivative as in the instantapplication). A panel of proteases that are capable of efficientlydigesting CD25 may be used as the catalytic domain.

[0253] In certain embodiments, the adzyme is an antagonist of IL-1. Suchadzymes can be used as part of a treatment for Rheumatoid arthritis. Thepathogenesis of RA is a complex process that leads to significant andchronic joint inflammation. Interleukin-1 (IL-1) is a central mediatorin RA and is a critical proinflammatory cytokine that has been found tobe abundant in the synovial fluid of RA patients. Kineret® (anakinra) isa recombinant, nonglycosylated form of the human interleukin 1 receptorantagonist (IL-iRa). Kineret® differs from native human IL-1Ra in thatit has the addition of a single methionine residue at its aminoterminus. Kineret® blocks the biologic activity of IL-1 by competitivelyinhibiting IL-1 binding to the interleukin-1 type I receptor (IL-1RI),which is expressed in a wide variety of tissues and organs. Therefore,Kineret® acts as a pure binder inhibitor. The subject adzyme, which canbe administered at a much lower dose because of its catalytic nature, isexpected to be a better therapeutic alternative. The adzyme addressdomain may use the same monoclonal antibody or functional derivativethereof (such as a Fab or scFv derivative as in the instantapplication). A panel of proteases that are capable of efficientlydigesting IL-1 may be used as the catalytic domain.

[0254] In certain embodiments, IgE (immunoglobulin E) may be the targetof an adzyme. IgE is a class of antibodies that protects the hostagainst invading parasites. IgE interacts with mast cells andeosinophils to protect the host against the invading parasite. TheIgE-immune cell complex is also responsible for many allergic orhypersensitivity reactions such as hay fever, asthma, hives andanaphylaxis. There are two major types of receptor for the Fc portion ofthe IgE on cells. A high affinity receptor is found primarily on mastcells and basophils. A low affinity receptor is found on CD23 cells. IgEattaches to these and acts as an antigen receptor. Xolair™ is ahumanized monoclonal antibody directed to the Fc portion of IgE andeffective in treating asthma. An adzyme targeted to and reducing theactivity of IgEs (generally by targeting the Fc portion) may be used totreat asthma. The adzyme address domain may use a monoclonal antibody orfunctional derivative thereof (such as a scFv derivative as in theinstant application), or a soluble ligand binding portion of an IgEreceptor. One or more of a panel of proteases that are capable ofefficiently digesting IgE may be used as the catalytic domain.

[0255] In certain embodiments, VEGF (Vascular Endothelial Growth Factor)may be the target of an adzyme. VEGF plays a critical role inangiogenesis (the formation of new blood vessels), particularly intumors and is also involved in the maintenance of established tumorblood vessels. VEGF is homodimeric and disulfide linked. Four humansplice variants of VEGF have been identified encoding, in the matureform, polypeptide monomers of 121, 165, 189, or 206 amino acids. Tworeceptor tyrosine kinases (RTKs), Flt-1 and FIk-1 bind VEGF with highaffinity. Avastin™ is an investigational recombinant humanized antibodyto VEGF, and shows effectiveness in improving the survival of metastaticcolorectal cancer patients. An adzyme targeted to and reducing theactivity of VEGF may be used to treat a variety of cancers, particularlycolorectal cancer. The adzyme address domain may use a monoclonalantibody or functional derivative thereof (such as a scFv derivative asin the instant application), or a soluble ligand binding portion of aVEGF receptor. One or more of a panel of proteases that are capable ofefficiently digesting VEGF may be used as the catalytic domain.

[0256] In certain embodiments, EGFR (Epidermal Growth Factor Receptor)may be the target for an adzyme. EGFR is expressed in a high percentageof many cancer types, including head and neck, colorectal, pancreatic,lung, esophageal, renal cell, prostate, bladder, cervical/uterus,ovarian and breast cancers. ERBITUX™ (formerly known as IMC-C225) is ahighly specific chimerized monoclonal antibody that binds to EGFR andblocks the ability of EGF to initiate receptor activation and signalingto the tumor. This blockade results in an inhibition of tumor growth byinterfering with the effects of EGFR activation including tumor invasionand metastases, cell repair and angiogenesis. ERBITUX™ has been used incombination with chemotherapy and radiation in animal models of humancancers. These preclinical findings indicate that when combined withchemotherapy or radiation, ERBITUX™ treatment provides an enhancedanti-tumor effect resulting in the elimination of tumors and thelong-term survival of the animals. An adzyme targeted to and reducingthe presence, ligand-binding or signaling capacity of EGFR may be usedto treat or prevent a variety of cancers, particularly colorectalcancer, and particularly when used in combination with one or moreadditional chemotherapeutic agents. The adzyme address domain may use amonoclonal antibody or functional derivative thereof (such as a scFvderivative as in the instant application), or a soluble ligand (such asEGF) for EGFR. One or more of a panel of proteases that are capable ofefficiently digesting extracellular portions of EGFR may be used as thecatalytic domain.

[0257] In certain embodiments, one or more alpha-4 integrins, such asbeta-1 and beta-7 may be the target(s) for an adzyme. Integrins aretransmembrane proteins, and the alpha-4-beta 1 (VLA-4) andalpha-4-beta-7 integrins help white blood cells, particularly Tlymphocytes and eosinophils, move from through the blood vessel wallsinto the tissues of the body at sites of inflammation, where these cellsthen participate in the inflammatory process. Antegren® is a humanizedmonoclonal antibody that binds to and blocks both the beta-1 and beta-7integrins, preventing the contribution of many cell types toinflammation; Antegren® shows effectiveness for treatment of Crohn'sdisease. An adzyme targeted to and reducing the presence orligand-binding capacity of these integrins may be used to treat orprevent a variety of inflammatory diseases, particularly Crohn'sdisease. The adzyme address domain may use a monoclonal antibody orfunctional derivative thereof (such as a scFv derivative as in theinstant application), or a soluble ligand for the targeted alpha-4integrins. One or more of a panel of proteases that are capable ofefficiently digesting extracellular portions of the targeted integrinsmay be used as the catalytic domain.

[0258] In certain embodiments, CCR-5 may be the target of an adzyme. Thehuman CCR5 chemokine receptor is a member of the rhodopsin superfamilyof G-linked receptors having seven hydrophobic transmembrane domains.CCR5 binds RANTES, MIP-1β and MIP-1α. Raport, C. J. et al. (1996) J.Biol. Chem. 271:17161. CCR5 facilitates infection by themacrophage-tropic HIV-1 virus, RANTES, MIP-11α and MIV-1β can suppressthe infection of susceptible cells by macrophage-tropic HIV-1 isolates.Choe, H. et al. (1996) Cell 85:1135. Cocchi, F. et al. (1995) Science270:1811. Although no CCR-5 targeted affinity agent has been approved,CCR-5 is implicated in HIV infection, and an adzyme targeted to andreducing the presence or HIV-binding capacity of CCR-5 may be used totreat or prevent asthma and other allergic reactions. The adzyme addressdomain may use a monoclonal antibody or functional derivative thereof(such as a scFv derivative as in the instant application), or a solubleligand for CCR-5. One or more of a panel of proteases that are capableof efficiently digesting extracellular portions of CCR-5 may be used asthe catalytic domain.

[0259] In certain embodiments, interleukin-4 may be the target of anadzyme. Human IL-4 is a pleiotropic cytokine produced by activated Tcells, mast cells, and basophils. The biological effects of IL-4 aremediated by the binding of IL-4 to specific cell surface receptors. Thefunctional high-affinity receptor for IL-4 includes a ligand bindingsubunit (IL-4 R) and a second subunit (β chain) that can modulate theligand binding affinity of the receptor complex. The gamma chain of the1L-2 receptor complex may also be a functional β chain of the IL-4receptor complex. Mature IL-4 is a 129 amino acid protein Yokota, T. etal., 1986, Proc. Natl. Acad. Sci. USA 83:5894. IL-4 activity may bemeasured, for example, in a cell proliferation assay employing a humanfactor-dependent cell line, TF-1. Kitamura et al., 1989 J. Cell Physiol.140:323. Although no IL-4 targeted affinity agent has been approved,IL-4 is implicated in allergies and asthma, and an adzyme targeted toand reducing the activity of IL-4 may be used to treat or prevent asthmaand other allergic reactions. The adzyme address domain may use amonoclonal antibody or functional derivative thereof (such as a scFvderivative as in the instant application), or a soluble ligand bindingportion of an IL-4 receptor. One or more of a panel of proteases thatare capable of efficiently digesting IL-4 may be used as the catalyticdomain.

[0260] In certain embodiments, IL-13 may be the target of an adzyme.Although no IL-13 targeted affinity agent has been approved, IL-13 iswidely recognized as a cytokine that is involved in asthma and variousallergies. Mature human IL-13 is a 112 amino acid polypeptide having asequence as described in GenBank accession no. P35225. McKenzie et al.1993, PNAS USA 90:3735-3739. IL-13 activity may be measured, forexample, in a cell proliferation assay employing a humanfactor-dependent cell line, TF-1. Kitamura et al., 1989 J. Cell Physiol.140:323. An adzyme targeted to and reducing the activity of IL13 may beused to treat or prevent asthma and other allergic reactions. The adzymeaddress domain may use a monoclonal antibody or functional derivativethereof (such as a scFv derivative as in the instant application), or asoluble ligand binding portion of an IL-13 receptor. One or more of apanel of proteases that are capable of efficiently digesting IL-13 maybe used as the catalytic domain.

[0261] (i) TNFα Antagonists

[0262] In certain embodiments, the subject adzyme is a TNFα antagonist,e.g., a “TNFα antagonist adzyme”. TNFα is a soluble homotrimer of 17 kDprotein subunits. A membrane-bound 26 kD precursor form of TNFα alsoexists. The pleiotropic activities of the potent proinflammatorycytokine TNF are mediated by two structurally related, but functionallydistinct, receptors, p55 and p75, that are coexpressed on most celltypes. To exert its biological activity, TNFα (a homotrimeric molecule)must bind to at least 2 cell surface receptors, causing cross-linkingand cell signaling. The majority of biologic responses classicallyattributed to TNFα are mediated by p55. In contrast, p75 has beenproposed to function as both a TNF antagonist by neutralizing TNFα andas a TNFα agonist by facilitating the interaction between TNFα and p55at the cell surface. The roles of p55 and p75 in mediating andmodulating the activity of TNFα in vivo have been examined in micegenetically deficient in these receptors. Selective deficits in severalhost defense and inflammatory responses are observed in mice lacking p55or both p55 and p75, but not in mice lacking p75. In these models, theactivity of p55 is not impaired by the absence of p75, arguing against aphysiologic role for p75 as an essential element of p55-mediatedsignaling. In contrast, exacerbated pulmonary inflammation anddramatically increased endotoxin induced serum TNFα levels in micelacking p75 suggest a dominant role for p75 in suppressing TNFα-mediatedinflammatory responses.

[0263] The p55 receptor (also termed TNF-R55, TNF-R1, or TNFRα) is a 55kd glycoprotein shown to transduce signals resulting in cytotoxic,antiviral, and proliferative activities of TNFα. The p75 receptor (alsotermed TNF-R75, TNF-R11, or TNFRα) is a 75 kDa glycoprotein that hasalso been shown to transduce cytotoxic and proliferative signals as wellas signals resulting in the secretion of GM-CSF. The extracellulardomains of the two receptors have 28% homology and have in common a setof four sub-domains defined by numerous conserved cysteine residues. Thep75 receptor differs, however, by having a region adjacent to thetransmembrane domain that is rich in proline residues and contains sitesfor O-linked glycosylation. Interestingly, the cytoplasmic domains ofthe two receptors share no apparent homology which is consistent withobservations that they can transduce different signals to the interiorof the cell.

[0264] To further illustrate, a TNFα antagonist adzyme can be directedto TNFα, e.g., in biological fluids, by way of one or more TNFαtargeting moieties. Exemplary TNFα targeting moieties include, but arenot limited to, the extracellular domains of TNFα receptors (orappropriate portions thereof), anti-TNFα antibodies or antigen bindingfragments thereof, or peptides or small molecules that (selectively)bind TNFα.

[0265] In certain preferred embodiments, the targeting moiety is derivedfrom the extracellular ligand binding domain of the p75 or p55 receptor,e.g., a portion sufficient to specifically bind to TNF-α. For instance,the targeting moiety can include a ligand binding fragment of p75, suchas from Leu23-Asp257 of the human p75 protein (SwissProt AccessionP20333) or a ligand binding fragment of p55, such as from Ile22-Thr211of the human p55 protein (Swiss-Prot Accession P19438). In certainembodiments, the targeting moiety of the subject adzymes can begenerated from Onercept (a fully human soluble fragment of p55) orEtanercept (Enbrel®, a dimeric construct in which two p75 extracellularfragments are linked to the Fc portion of human IgG1).

[0266] In other preferred embodiments, the targeting moiety is derivedfrom an antibody that binds to TNFα, or an antigen binding domainthereof. For instance, the subject adzymes can generated using themonoclonal anti-TNFα antibody is infliximab (Remicade®), or the variabledomains of one or both of the heavy and light chains thereof, such asthe Fv fragment. Infliximab is a chimeric human/mouse monoclonalanti-TNFα antibody composed of the constant regions of human (Hu) IgGIκ,coupled to the Fv region of a high-affinity neutralizing murineanti-HuTNFα antibody. Likewise, the subject adzyme can including atargeting moiety derived from the human anti-TNF antibody D2E7, alsoknown as adalumimab.

[0267] In still other embodiments, the TNFα targeting moiety is apeptide. For instance, Guo et al. (2002) Di Yi Jun Yi Da Xue Xue Bao.22(7):597 describes the screening of TNFα-binding peptides by phagedisplay. That reference teaches a number of short peptides that could beused to generate TNFα-targeted adzymes. Merely to illustrate, the TNFαtargeting moiety can be a peptide having the sequence ALWHWWH or(T/S)WLHWWA.

[0268] The ability of any particular adzyme to act alter the activity ofTNFα can be assayed using any of a variety of cell-based and cell-freeassay systems well known in the art. Exemplary assays include, but arenot limited to, L929 assay, endothelial procoagulation assays, tumorfibrin deposition assays, cytotoxicity assay, tumor regression assays,receptor binding assays, arthritic index assays in mouse model systems,and the like. In certain preferred embodiments of TNFα antagonistadzymes, their biological activities will include one or more of:inhibition of TNF-α cytotoxicity in L929 cells; blocking ofprostaglandin E2 production and expression of cell-associated IL1 byhuman dermal fibroblasts; blocking of TNF-α binding to the promonocyticcell line U937; blocking of TNF-α induced respiratory burst in humanneutrophils; blocking of TNF-stimulated neutrophil lucigenin-dependentchemiluminescence response and superoxide formation; significantlyreducing the priming ability of TNF-α for a response to the chemotacticpeptide fMLP; blocking of class I antigen expression in the human Colo205 tumor cell line; affecting TNF-α synergism with HLA-DR antigenexpression induced by IFN-γ (yet preferably having no effect on IFN-γactivity).

[0269] In certain embodiments, the TNFα antagonist adzyme will modifythe substrate TNFα protein in a manner that produces a product that isitself an antagonist of TNFα. For instance, the adzyme can include acatalytic domain that cleaves a site in the TNFα polypeptide to producea product that retains the ability to bind, for example, to the p55receptor but with a greatly reduced ability to activate the receptor(e.g., has an impaired ability to induce a cytotoxic response) so as tobe an antagonist of native TNFα. For instance, the cleavage product mayretain the ability to interact with native TNFα to form stable mixedtrimers that bind to receptors but are incapable of activating receptorsignaling. To further illustrate, the sites within the human TNF-αmolecule that can be targeted for cleavage by an adzyme may be locatedat or near residues 29 to 34, 71-73, 86, and 143-146. For instance, acatalytic domain having a trypsin-like specificity can be used in anadzyme that selectively cleaves Arg⁴⁴ of human TNFα. Likewise, an adzymeincluding the catalytic domain of granzyme B can be used to targetAsp¹⁴³. Residues within these regions are believed to be important forTNF-α cytotoxic activity. Adzyme cleavage products having thecombination of antagonist activity and reduced cytotoxicity can beidentified by the screening assays described above.

[0270] The subject TNFα antagonist adzymes can be used to treat variousTNF-associated disorders, e.g., disorders or diseases that areassociated with, result from, and/or occur in response to, elevatedlevels of TNFα. Such disorders may be associated with episodic orchronic elevated levels of TNFα activity and/or with local or systemicincreases in TNFα activity. Such disorders include, but are not limitedto, inflammatory diseases, such as arthritis and inflammatory boweldisease, and congestive heart failure.

[0271] TNFα causes pro-inflammatory actions which result in tissueinjury, such as degradation of cartilage and bone, induction of adhesionmolecules, inducing procoagulant activity on vascular endothelial cells,increasing the adherence of neutrophils and lymphocytes, and stimulatingthe release of platelet activating factor from macrophages, neutrophilsand vascular endothelial cells. In certain preferred embodiments, theTNFα antagonist adzyme reduces the inflammatory activity of TNFα.

[0272] Recent evidence also associates TNFα with infections, immunedisorders, neoplastic pathologies, autoimmune pathologies andgraft-versus-host pathologies. For instance, TNFα is understood to playa central role in gram-negative sepsis and endotoxic shock, includingfever, malaise, anorexia, and cachexia. Endotoxin strongly activatesmonocyte/macrophage production and secretion of TNFα and other cytokines(Kombluth et al., J. Immunol. 137:2585-2591 (1986)). Circulating TNFαlevels increase in patients suffering from gram-negative sepsis. Thus,the subject TNFα antagonist adzymes may used as part of a treatmentprotocol for inflammatory diseases, autoimmune diseases, viral,bacterial and parasitic infections, malignancies, and neurogenerativediseases, such as for therapy in rheumatoid arthritis and Crohn'sdisease.

[0273] There is evidence that TNFα is also involved in cachexia incancer, infectious pathology, and other catabolic states. Accordingly,the TNFα antagonist adzymes can also be used to reduce muscle wastingassociated with such disorders, or any other in which cachexia is anissue in patient management.

[0274] Accordingly, the present invention provides methods in which thesubject adzymes can be used as part of treatments for modulating orreducing the severity of at least one immune related disease, in a cell,tissue, organ, animal, or patient including, but not limited to, atleast one of rheumatoid arthritis, juvenile rheumatoid arthritis,systemic onset juvenile rheumatoid arthritis, psoriatic arthritis,ankylosing spondilitis, gastric ulcer, seronegative arthropathies,osteoarthritis, inflammatory bowel disease, ulcerative colitis, systemiclupus erythematosis, antiphospholipid syndrome,iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary fibrosis,systemic vasculitis/wegener's granulomatosis, sarcoidosis,orchitis/vasectomy reversal procedures, allergic/atopic diseases,asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergicconjunctivitis, hypersensitivity pneumonitis, transplants, organtransplant rejection, graft-versus-host disease, systemic inflammatoryresponse syndrome, sepsis syndrome, gram positive sepsis, gram negativesepsis, culture negative sepsis, fungal sepsis, neutropenic fever,urosepsis, meningococcemia, trauma/hemorrhage, bums, ionizing radiationexposure, acute pancreatitis, adult respiratory distress syndrome,rheumatoid arthritis, alcohol-induced hepatitis, chronic inflammatorypathologies, sarcoidosis, Crohn's pathology, sickle cell anemia,diabetes, nephrosis, atopic diseases, hypersensitity reactions, allergicrhinitis, hay fever, perennial rhinitis, conjunctivitis, endometriosis,asthma, urticaria, systemic anaphalaxis, dermatitis, pernicious anemia,hemolytic disesease, thrombocytopenia, graft rejection of any organ ortissue, kidney translplant rejection, heart transplant rejection, livertransplant rejection, pancreas transplant rejection, lung transplantrejection, bone marrow transplant (BMT) rejection, skin allograftrejection, cartilage transplant rejection, bone graft rejection, smallbowel transplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions, Gravesdisease, Raynoud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, antibody-meditated cytotoxicity, type IIIhypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), polyneuropathy, organomegaly,endocrinopathy, monoclonal gammopathy, skin changes syndrome,antiphospholipid syndrome, pemphigus, scleroderma, mixed connectivetissue disease, idiopathic Addison's disease, diabetes mellitus, chronicactive hepatitis, primary billiary cirrhosis, vitiligo, vasculitis,post-MI cardiotomy syndrome, type IV hypersensitivity, contactdermatitis, hypersensitivity pneumonitis, allograft rejection,granulomas due to intracellular organisms, drug sensitivity,metabolic/idiopathic, Wilson's disease, hemachromatosis,alpha-1-antitrypsin deficiency, diabetic retinopathy; Hashimoto'sthyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axisevaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis,cachexia, cystic fibrosis, neonatal chronic lung disease, chronicobstructive pulmonary disease (COPD), familial hematophagocyticlymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,nephrotic syndrome, nephritis, glomerular nephritis, acute renalfailure, hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy,anti-cd3 therapy, cytokine therapy, chemotherapy, radiation therapy(e.g., including but not limited to asthenia, anemia, cachexia, and thelike), chronic salicylate intoxication, and the like.

[0275] In one embodiment, a TNFα adzyme is used to treathypergastrinemia, such as Helicobacter Pylori-induced gastritis.

[0276] The present invention also provides methods for using the subjectTNFα antagonist adzymes for modulating or treating at least onecardiovascular disease in a cell, tissue, organ, animal, or patient,including, but not limited to, at least one of cardiac stun syndrome,myocardial infarction, congestive heart failure, stroke, ischemicstroke, hemorrhage, arteriosclerosis, atherosclerosis, restenosis,diabetic ateriosclerotic disease, hypertension, arterial hypertension,renovascular hypertension, syncope, shock, syphilis of thecardiovascular system, heart failure, cor pulmonale, primary pulmonaryhypertension, cardiac arrhythmias, atrial ectopic beats, atrial flutter,atrial fibrillation (sustained or paroxysmal), post perfusion syndrome,cardiopulmonary bypass inflammation response, chaotic or multifocalatrial tachycardia, regular narrow QRS tachycardia, specific arrythmias,ventricular fibrillation, His bundle arrhythmias, atrioventricularblock, bundle branch block, myocardial ischemic disorders, coronaryartery disease, angina pectoris, myocardial infarction, cardiomyopathy,dilated congestive cardiomyopathy, restrictive cardiomyopathy, valvularheart diseases, endocarditis, pericardial disease, cardiac tumors,aordic and peripheral aneuryisms, aortic dissection, inflammation of theaorta, occulsion of the abdominal aorta and its branches, peripheralvascular disorders, occulsive arterial disorders, peripheralatherlosclerotic disease, thromboangitis obliterans, functionalperipheral arterial disorders, Raynaud's phenomenon and disease,acrocyanosis, erythromelalgia, venous diseases, venous thrombosis,varicose veins, arteriovenous fistula, lymphedema, lipedema, unstableangina, reperfusion injury, post pump syndrome, ischemia-reperfusioninjury, and the like.

[0277] The present invention also provides methods using the subjectTNFα antagonist adzymes for modulating or treating at least oneinfectious disease in a cell, tissue, organ, animal or patient,including, but not limited to, at least one of: acute or chronicbacterial infection, acute and chronic parasitic or infectiousprocesses, including bacterial, viral and fungal infections, HIVinfection/HIV neuropathy, meningitis, hepatitis (A, B or C, or thelike), septic arthritis, peritonitis, pneumonia, epiglottitis, E. coliinfection, hemolytic uremic syndrome/thrombolytic thrombocytopenicpurpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy,toxic shock syndrome, streptococcal myositis, gas gangrene,mycobacterium tuberculosis, mycobacterium avium intracellulare,pneumocystis carinii pneumonia, pelvic inflammatory disease,orchitis/epidydimitis, legionella, lyme disease, influenza a,epstein-barr virus, vital-associated hemaphagocytic syndrome, vitalencephalitis/aseptic meningitis, and the like.

[0278] The present invention also provides methods for modulating ortreating at least one malignant disease in a cell, tissue, organ, animalor patient, including, but not limited to, at least one of: leukemia,acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell orFAB ALL, acute myeloid leukemia (AML), chromic myelocytic leukemia(CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia,myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, amalignamt lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, multiplemyeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma,nasopharyngeal carcinoma, malignant histiocytosis, paraneoplasticsyndrome/hypercalcemia of malignancy, solid tumors, adenocarcinomas,sarcomas, malignant melanoma, hemangioma, metastatic disease, cancerrelated bone resorption, cancer related bone pain, and the like.

[0279] The present invention also provides methods that use TNFαantagonist adzymes for modulating or treating at least one neurologicdisease in a cell, tissue, organ, animal or patient, including, but notlimited to, at least one of: neurodegenerative diseases, multiplesclerosis, migraine headache, AIDS dementia complex, demyelinatingdiseases, such as multiple sclerosis and acute transverse myelitis;extrapyramidal and cerebellar disorders' such as lesions of thecorticospinal system; disorders of the basal ganglia or cerebellardisorders; hyperkinetic movement disorders such as Huntington's Choreaand senile chorea; drug-induced movement disorders, such as thoseinduced by drugs which block CNS dopamine receptors; hypokineticmovement disorders, such as Parkinson's disease; Progressive supranucleoPalsy; structural lesions of the cerebellum; spinocerebellardegenerations, such as spinal ataxia, Friedreich's ataxia, cerebellarcortical degenerations, multiple systems degenerations (Mencel,Dejerine-Thomas, Shi-Drager, and Machado-Joseph); systemic disorders(Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, andmitochondrial multi.system disorder); demyelinating core disorders, suchas multiple sclerosis, acute transverse myelitis; and disorders of themotor unit such as neurogenic muscular atrophies (anterior horn celldegeneration, such as amyotrophic lateral sclerosis, infantile spinalmuscular atrophy and juvenile spinal muscular atrophy); Alzheimer'sdisease; Down's Syndrome in middle age; Diffuse Lewy body disease;Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronicalcoholism; Creutzfeldt-Jakob disease; Subacute sclerosingpanencephalitis, Hallerrorden-Spatz disease; and Dementia pugilistica,and the like.

[0280] The TNFα antagonist adzymes can be administered before,concurrently, and/or after (referred to herein as “concomitantly with”)other drugs, such as at least one selected from an antirheumatic (e.g.,methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, goldsodium thiomalate, hydroxychloroquine sulfate, leflunomide,sulfasalzine), a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anethetic, a neuromuscular blocker, an antimicrobial (e.g.,aminoglycoside, an antifungal, an antiparasitic, an antiviral, acarbapenem, cephalosporin, a flurorquinolone, a macrolide, a penicillin,a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic,a corticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, domase alpha (Pulmozyrne), a cytokine or acytokine antagonist.

[0281] The subject adzymes can also be administered concomitantly withcompounds that prevent and/or inhibit TNF receptor signaling, such asmitogen activated protein (MAP) kinase inhibitors; compounds which blockand/or inhibit membrane TNF cleavage, such as metalloproteinaseinhibitors; compounds which block and/or inhibit TNF activity, such asangiotensin converting enzyme (ACE) inhibitors (e.g., captopril); andcompounds which block and/or inhibit TNF production and/or synthesis,such as MAP kinase inhibitors.

[0282] (ii) IL-1b Antagonists

[0283] In certain embodiments, the subject adzyme is an Interleukin-1antagonist, e.g., an “IL-1 antagonist adzyme”. Interleukin-1 is amulti-functional proinflammatory cytokine that mediates innate andadaptive immune responses in multiple cell types. It is believed to playa role in numerous diseases including arthritis, asthma/allergy,osteoporosis, and stroke (for review, see Dinarello (1998) Int. Rev.Immunol. 16, 457-499). The IL-1 family actually consists of two proteinswith similar biological activity, IL-1α and IL-1β, as well as anonsignaling ligand termed the IL-1 receptor antagonist (IL-1ra). Allthree proteins exhibit a similar tertiary structure comprised of 12β,strands that make up a barrel-shaped β-trefoil with pseudo-3-foldsymmetry. IL-1β, is thought to be the primary circulating cytokine thatmediates the systemic effects of IL-1.

[0284] IL-1 exerts its biological action by binding and activating themembrane-associated IL1R-I. A second receptor, termed the IL-1Raccessory protein (AcP), is not involved in direct ligand binding but isrequired for IL-1 signal transduction by complexing with IL-1 and theIL1R-I. IL1R-I and AcP both contain extracellular portions with threeIg-like domains and cytoplasmic portions containing conserved signalingmotifs. A third IL-1 receptor exists termed the type II IL-1R (IL1R-II)that has a extracellular structure similar to that of IL1R-I and AcP butthat contains a truncated cytoplasmic tail incapable of signaling. Thisreceptor acts as a decoy by binding IL-1 with high affinity andneutralizing its activity. IL1R-II can also be proteolytically cleaved,which releases the extracellular domain from the cell surface. Thiscreates a soluble form of the receptor (sIL1R-II) that possesses highaffinity for IL-1β, but only low affinity for IL-1α, and virtually noaffinity for IL-Ira.

[0285] In certain preferred embodiments, the subject adzymes are IL-1antagonist adzyme that act on IL-1, particularly IL-1β, present inbiological fluids. Exemplary IL-1 targeting moieties that be adapted foruse in such adzymes include, but are not limited to, the extracellulardomains of IL-1 receptors or appropriate portions thereof, IL1R-II or aportion thereof, anti-IL-1 antibodies or antigen binding fragmentsthereof, or peptides or small molecules that (selectively) bind IL-1.

[0286] In certain preferred embodiments, the targeting moiety is derivedfrom IL1R-II, e.g., a portion sufficient to specifically bind to Il1β.For instance, the targeting moiety can include a ligand binding domainfrom IL1R-II from the human IL1R-II protein (GI Accession 640248, PR1Accession 2IRT_A).

[0287] The inhibitory activity of an IL-1 antagonist adzyme can beassayed using any of a variety of cell-based and cell-free assay systemswell known in the art. For instance, IL-1 antagonist adzymes can beidentified using the mixed lymphocyte response (MLR) andphytohemagglutinin A (PHA) assay, which is useful for identifying immunesuppressive molecules in vitro that can be used for treatinggraft-versus-host disease. The results obtained from these assays aregenerally predictive of their in vivo effectiveness.

[0288] Another assay that be used to assess the adzyme is with respectto inhibition of immune responsiveness involves the mitogenicstimulation of lymphocytes with mitogenic substances of plant origin.The most widely used plant molecule is PHA. Although PHA stimulates DNAsynthesis non-specifically in a large number of lymphocytes, unlike trueantigenic stimulation which causes mitogenesis of sub-populations oflymphocytes, the susceptibility of a patient's lymphocytes to PHAstimulation has been shown to correlate with the overall immuneresponsiveness of the patient.

[0289] Thus, it will be appreciated as to both the mixed lymphocyte andPHA assay that they are valuable for identifying immune suppressive IL-1antagonist adzymes.

[0290] In addition to the above immunosuppressive assays, a secondarymixed lymphocyte reaction assay may also be used. The secondary mixedlymphocyte assays differs from the primary mixed lymphocyte reactionassays in that they employ many more primed responder cells that areresponsive to the primary stimulating cells. The presence of suchresponsive cells is a reflection of immunological memory in an ongoingimmunological response. The protocol for carrying out a secondary mixedlymphocyte assay involves performing a primary lymphocyte assay asdescribed above, and recovering viable cells about 9-10 days after theprimary mixed lymphocyte reaction exhibits little or no cellproliferation. Generally between 10% to 50% of the original input cellsare recovered in viable condition. These cells are then used in thesecondary mixed lymphocyte reaction.

[0291] The subject adzymes can also be assessed for their ability toblock IL-1 mediated cytokine production. Assays for cytokine productionand/or proliferation of spleen cells, lymph node cells or thymocytes arewell known in the art.

[0292] In still other embodiments, the subject adzymes can be assessedfor their effect on proliferation and differentiation of hematopoieticand lymphopoietic cells.

[0293] In certain embodiments, the IL-1 antagonist adzyme will modifythe substrate IL-1β protein in a manner that produces a product that isitself an antagonist of IL1β. For instance, the adzyme can include acatalytic domain that cleaves a site in the IL-1β polypeptide to producea product that retains the ability to bind, for example, to the IL1receptor but with a greatly reduced ability to activate the receptor soas to be an antagonist of native Il-1β. To further illustrate, theArg¹²⁷ residue of human IL-1β can be targeted for cleavage by an adzymehaving a catalytic domain with trypsin-like specificity. Mutation ofArg¹²⁷ has been demonstrated to reduce the bioactivity of IL-1β greatlywhile only having slight effect on receptor binding affinity. The IL-1mediated diseases which may be treated or prevented by the IL-1antagonist adzymes of this invention include, but are not limited to,inflammatory diseases, autoimmune diseases, proliferative disorders,infectious diseases, and degenerative diseases. The apoptosis-mediateddiseases which may be treated or prevented by the IL-1 antagonistadzymes of this invention include degenerative diseases.

[0294] Inflammatory diseases which may be treated or prevented include,but are not limited to osteoarthritis, acute pancreatitis, chronicpancreatitis, asthma, and adult respiratory distress syndrome.Preferably the inflammatory disease is osteoarthritis or acutepancreatitis.

[0295] Autoimmune diseases which may be treated or prevented include,but are not limited to, glomeralonephritis, rheumatoid arthritis,systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves'disease, autoimmune gastritis, insulin-dependent diabetes mellitus (TypeI), autoimmune hemolytic anemia, autoimmune neutropenia,thrombocytopenia, chronic active hepatitis, myasthenia gravis, multiplesclerosis, inflammatory bowel disease, Crohn's disease, psoriasis, andgraft vs. host disease. Preferably the autoimmune disease is rheumatoidarthritis, inflammatory bowel disease, Crohn's disease, or psoriasis,Destructive bone disorders which may be treated or prevented include,but are not limited to, osteoporosis and multiple myeloma-related bonedisorder.

[0296] Proliferative diseases which may be treated or prevented include,but are not limited to, acute myelogenous leukemia, chronic myelogenousleukemia, metastatic melanoma, Kaposi's sarcoma, and multiple myeloma.

[0297] Infectious diseases which may be treated or prevented include,but are not limited to, sepsis, septic shock, and Shigellosis.

[0298] The IL-1-mediated degenerative or necrotic diseases which may betreated or prevented by the IL-1 antagonist adzymes of this inventioninclude, but are not limited to, Alzheimer's disease, Parkinson'sdisease, cerebral ischemia, and myocardial ischemia. Preferably, thedegenerative disease is Alzheimer's disease.

[0299] The apoptosis-mediated degenerative diseases which may be treatedor prevented by the IL-1 antagonist adzymes of this invention include,but are not limited to, Alzheimer's disease, Parkinson's disease,cerebral ischemia, myocardial ischemia, spinal muscular atrophy,multiple sclerosis, AIDS-related encephalitis, HIV-related encephalitis,aging, alopecia, and neurological damage due to stroke.

[0300] (e) Biomolecular Targets in Non-Therapeutic Contexts

[0301] Adzymes may be used in a number of non-medical applications,including but are not limited to, agriculture, environmental protection,food etc., and such adzymes will be targeted accordingly.

[0302] Adzymes may be used to upgrade nutritional quality and removinganti-nutritional factors from feed components, such as barley- andwheat-based feeds. Targets for such adzymes may include gluten meal,fiber, prions (e.g., PrP, the causative agent for bovine spongiformencephalopathy), dioxin, pesticides, herbicides, starches, lipids,cellulose, pectin, certain sugars (e.g., lactose, maltose) andpolysaccharides.

[0303] Adzyme may be used in industrial processes such as wasteprocessing, textile manufacture or paper production, or essentially anyother process that employs an enzyme, where the enzyme can be replacedby an adzyme with improved effectiveness. Examples of targets for suchapplications include cellulose, hemicellulose, pectin, lignin, starch,peroxides, phosphates and nitrates.

[0304] Adzyme may be used in detergents or other cleaning agents,providing targeted elimination of selected soils or stains. Targets forsuch adzymes may include chlorophyll, hemoglobin, heme groups,hydrocarbons, avidin, ovalbumin, and various pigments and dyes.

[0305] Adzymes may be used for the cleanup of various environmentalcontaminants, such oil, pesticides, herbicides and waste products fromchemical manufacture. Targets for such adzymes include hydrocarbons,halogenated hydrocarbons (particularly halogenated hydrocarbonscontaining aromatic moieties), cyanides, carbon monoxide, nitrousoxides, heavy metals, organometallic compounds, organophosphates andcarbamates.

[0306] B. Exemplary Catalytic Domains

[0307] As used herein, the term “catalytic domain” includes any moietycapable of acting on a target to induce a chemical change, therebymodulate its activity, i.e., a moiety capable of catalyzing a reactionwithin a target. The catalytic domain may be a naturally occurringenzyme, a catalytically active fragment thereof, or an engineeredenzyme, e.g., a protein engineered to have an enzymatic activity, suchas a protein designed to contain a serine protease active motif. Acatalytic domain need comprise only the arrangement of amino acids thatare effective to induce the desired chemical change in the target. Theymay be N-terminal or C-terminal truncated versions of natural enzymes,mutated versions, zymogens, or complete globular domains. The catalyticdomain may be a non protein physiologically compatible catalyst.

[0308] The catalytic domain may comprise an enzymatically active sitethat alone is promiscuous, binding with a vulnerable site it recognizeson many different biomolecules, and may have relatively poor reactionkinetics. Both of these features are normally antithetical to sound drugdevelopment, but often are desireable in adzyme constructs, where theaddress specifies preference for the desired targeted biomolecule, andits binding properties often dominate kinetics, i.e., assurepreferential collision between the catalytically active site and thetarget.

[0309] The catalytic domain also may be a protein that modifies thetarget so that it is recognized and acted upon by another enzyme (e.g.,an enzyme that is already present in a subject). In another embodiment,the catalytic domain may be a moiety that alters the structure of thetarget so that its activity is inhibited or upregulated. Many naturallyoccurring enzymes activate other enzymes, and these can be exploited inaccordance with the invention.

[0310] The catalytic moiety of the adzyme can be a protease, aglycosidase, a lipase, or other hydrolases, or other enzymatic activity,including isomerases, transferases (including kinases), lyases,oxidoreductases, oxidases, aldolases, ketolases, glycosidases,transferases and the like.

[0311] Other potentially useful enzymes include oxidoreductases such asthose acting on groups donating CH—OH, aldehyde or oxo, CH—CH, CH—NH₂,CH—NH, NADH or NADPH, other nitrogenous compounds, sulfur, heme,diphenols, hydrogen, single donors with incorporation of molecularoxygen, paired donors, —CH₂ groups, reduced flavodoxin, iron-sulfurproteins, and oxidizing metal ions, and those acting on a superoxideradicals or peroxide as acceptors (peroxidases). The term“oxidoreductase” encompasses enzymes which catalyze the reduction oroxidation of a molecule. Examples of oxidoreductases includedehydrogenases, reductases, oxidases, oxygenases, hydrolases, andperoxidases.

[0312] Transferases include enzymes that catalyze the transfer of agroup of atoms from one molecule to another. Useful transferasecatalytic domains transfer carbon groups, nitrogenous groups,phosphorous-containing groups, sulfur-containing groups,seleniumcontaining groups, or aldehyde or ketone residues, and includeacyltransferases or glycosyltransferases transferring alkyl or arylgroups. Examples of transferases include aminotransferases, kinases, andmyristolases.

[0313] Other potentially useful enzymes include carbon-carbon,carbon-oxygen, carbonnitrogen, carbon-sulfur carbon-halide andphosphorus-oxygen lyases. Lyases add a small molecule to a double bond.Examples of lyases include synthases, decarboxylases and dehydratases.

[0314] The adzyme can include a ligase domain, e.g., a catalytic domainthat catalyzes the formation of carbon-oxygen bonds, carbon-sulfurbonds, carbon-nitrogen bonds, carboncarbon bonds, and phosphoric esterbonds. Ligases catalyze bond forming reactions which join together twoor more molecules. Examples of ligases include carboxylases andsynthetases.

[0315] Isomerases, racemases, epimerases, cis-trans-isomerases,intramolecular oxidoreductases, intramolecular transferases (mutases),intramolecular lyases also are potentially useful.

[0316] Glycosidases can also be a source for the catalytic domain of anadzyme. These enzymes are defined as glycolytic enzymes which can alterthe carbohydrate structure of a protein substrate, and may be useful ininstances such as when carbohydrate-mediated interaction of the targetedsubstrate with other proteins (such as a ligand-receptor interaction) isimportant to biological activity, or where the carbohydrate influencesthe half-life or biodistribution of the targeted substrate. Lysozyme isan example of a hydrolytic enzyme directed to polysaccharides.

[0317] Likewise, lipases can be employed in adzymes that alter membranestructure.

[0318] Many examples of each type of enzyme have been identified and canbe found in public databases, e.g., SwissProt, PIR, PRF, or the databasemaintained by the National Centers for Biotechnology Information (NCBI).Further, high resolution three dimensional structural coordinates formany enzymes can be found in the database maintained by the ResearchCollaboratory for Structural Bioinformatics (RCSB). The unresolvedstructures of proteins often can be predicted using a technique known asthreading. Threading algorithms are described in the literature and canbe found in Alexandrov N. N., et al., (1998) Bioinformatics 14:206-11,Labesse G, et al. (1997) Proteins 1:38-42, Xu, Y. et al. (1999). ProteinEng., 12: 899-907, Russel A. J., et al. (2002) Proteins 47:496-505, andReva, B., et al. (2002) Proteins 47:180-93.

[0319] In a preferred embodiment, the catalytic domain is a protease.Examples of proteases, or catalytically active fragments thereof, thatcan be utilized to this end include serine proteases, cysteineproteases, aspartate or acid proteases, metalloproteases or any otherprotease capable of cleaving the amide backbone of the targetedsubstrate.

[0320] In certain preferred embodiments, the subject adzyme incorporatesa peptidase catalytic activity, e.g., such as may be derived using anenzyme which is designates by the International Union of Biochemistryand Molecular Biology (1984) as subclass E.C 3.4-.-. For example, thesubject method can be used to determine the specificity of anaminopeptidase (EC 3.4.11.-), a dipeptidase (EC 3.4.13.-), adipeptidyl-peptidase or tripeptidyl peptidase (EC 3.4.14.-), apeptidyl-dipeptidase (EC 3.4.15.-), a serine-type carboxypeptidase (EC3.4.16.-), a metallocarboxypeptidase (EC 3.4.17.-), a cysteine-typecarboxypeptidase (EC 3.4.18.-), an omegapeptidase (EC 3.4.19.-), aserine proteinase (EC 3.4.21.-), a cysteine proteinase (EC 3.4.22.-), anaspartic proteinase (EC 3.4.23.-), a metallo proteinase (EC 3.4.24.-),or a proteinase of unknown mechanism (EC 3.4.99.-). Exemplary peptidehydrolyases which can be adapted for use in the subject adzymes include:3.4.11.1 Leucyl aminopeptidase. 3.4.11.2 Membrane alanineaminopeptidase. 3.4.11.3 Cystinyl aminopeptidase. 3.4.11.4 Tripeptideaminopeptidase. 3.4.11.5 Prolyl aminopeptidase. 3.4.11.6 AminopeptidaseB. 3.4.11.7 Glutamyl aminopeptidase. 3.4.11.9 Xaa-Pro aminopeptidase.3.4.11.10 Bacterial leucyl aminopeptidase. 3.4.11.13 Clostridialaminopeptidase. 3.4.11.14 Cytosol alanyl aminopeptidase. 3.4.11.15 Lysylaminopeptidase. 3.4.11.16 Xaa-Trp aminopeptidase. 3.4.11.17 Tryptophanylaminopeptidase. 3.4.11.18 Methionyl aminopeptidase. 3.4.11.19D-stereospecific aminopeptidase. 3.4.11.20 Aminopeptidase Ey. 3.4.11.22Vacuolar aminopeptidase I. 3.4.13.3 Xaa-His dipeptidase. 3.4.13.4Xaa-Arg dipeptidase. 3.4.13.5 Xaa-methyl-His dipeptidase. 3.4.13.6Cys-Gly dipeptidase. 3.4.13.7 Glu-Glu dipeptidase. 3.4.13.8 Pro-Xaadipeptidase. 3.4.13.9 Xaa-Pro dipeptidase. 3.4.13.12 Met-Xaadipeptidase. 3.4.13.17 Non-stereospecific dipeptidase. 3.4.13.18 Cytosolnon-specific dipeptidase. 3.4.13.19 Membrane dipeptidase. 3.4.13.20Beta-Ala-His dipeptidase. 3.4.14.1 Dipeptidyl-peptidase I. 3.4.14.2Dipeptidyl-peptidase II. 3.4.14.4 Dipeptidyl-peptidase III. 3.4.14.5Dipeptidyl-peptidase IV. 3.4.14.6 Dipeptidyl-dipeptidase. 3.4.14.9Tripeptidyl-peptidase I. 3.4.14.10 Tripeptidyl-peptidase II. 3.4.14.11Xaa-Pro dipeptidyl-peptidase. 3.4.15.1 Peptidyl-dipeptidase A. 3.4.15.4Peptidyl-dipeptidase B. 3.4.15.5 Peptidyl-dipeptidase Dcp. 3.4.16.2Lysosomal Pro-X carboxypeptidase. 3.4.16.4 Serine-type D-Ala-D-Alacarboxypeptidase. 3.4.16.5 Carboxypeptidase C. 3.4.16.6 CarboxypeptidaseD. 3.4.17.1 Carboxypeptidase A. 3.4.17.2 Carboxypeptidase B. 3.4.17.3Lysine(arginine) carboxypeptidase. 3.4.17.4 Gly-X carboxypeptidase.3.4.17.6 Alanine carboxypeptidase. 3.4.17.7 Transferred entry:3.4.19.10. 3.4.17.8 Muramoylpentapeptide carboxypeptidase. 3.4.17.10Carboxypeptidase H. 3.4.17.11 Glutamate carboxypeptidase. 3.4.17.12Carboxypeptidase M. 3.4.17.13 Muramoyltetrapeptide carboxypeptidase.3.4.17.14 Zinc D-Ala-D-Ala carboxypeptidase. 3.4.17.15 CarboxypeptidaseA2. 3.4.17.16 Membrane Pro-X carboxypeptidase. 3.4.17.17 Tubulinyl-Tyrcarboxypeptidase. 3.4.17.18 Carboxypeptidase T. 3.4.17.19 Thermostablecarboxypeptidase 1. 3.4.17.20 Carboxypeptidase U. 3.4.17.21 Glutamatecarboxypeptidase II. 3.4.17.22 Metallocarboxypeptidase D. 3.4.18.1Cysteine-type carboxypeptidase. 3.4.19.1 Acylaminoacyl-peptidase.3.4.19.2 Peptidyl-glycinamidase. 3.4.19.3 Pyroglutamyl-peptidase I.3.4.19.5 Beta-aspartyl-peptidase. 3.4.19.6 Pyroglutamyl-peptidase II.3.4.19.7 N-formylmethionyl-peptidase. 3.4.19.8Pteroylpoly-gamma-glutamate carboxypeptidase. 3.4.19.9 Gamma-glutamylhydrolase. 3.4.19.11 Gamma-D-glutamyl-meso-diaminopimelate peptidase I.3.4.21.1 Chymotrypsin. 3.4.21.2 Chymotrypsin C. 3.4.21.3 Metridin.3.4.21.4 Trypsin. 3.4.21.5 Thrombin. 3.4.21.6 Coagulation factor Xa.3.4.21.7 Plasmin. 3.4.21.8 Transferred entry: 3.4.21.34 and 3.4.21.35.3.4.21.9 Enteropeptidase. 3.4.21.10 Acrosin. 3.4.21.11 Transferredentry: 3.4.21.36 and 3.4.21.37. 3.4.21.12 Alpha-lytic endopeptidase.3.4.21.19 Glutamyl endopeptidase. 3.4.21.20 Cathepsin G. 3.4.21.21Coagulation factor VIIa. 3.4.21.22 Coagulation factor IXa. 3.4.21.25Cucumisin. 3.4.21.26 Prolyl oligopeptidase. 3.4.21.27 Coagulation factorXIa. 3.4.21.32 Brachyurin. 3.4.21.34 Plasma kallikrein. 3.4.21.35 Tissuekallikrein. 3.4.21.36 Pancreatic elastase. 3.4.21.37 Leukocyte elastase.3.4.21.38 Coagulation factor XIIa. 3.4.21.39 Chymase. 3.4.21.41Complement component Clr. 3.4.21.42 Complement component Cls. 3.4.21.43Classical-complement pathway C3/C5 convertase. 3.4.21.45 Complementfactor I. 3.4.21.46 Complement factor D. 3.4.21.47Alternative-complement pathway C3/C5 convertase. 3.4.21.48 Cerevisin.3.4.21.49 Hypodermin C. 3.4.21.50 Lysyl endopeptidase. 3.4.21.53Endopeptidase La. 3.4.21.54 Gamma-renin. 3.4.21.55 Venombin AB.3.4.21.57 Leucyl endopeptidase. 3.4.21.59 Tryptase. 3.4.21.60Scutelarin. 3.4.21.61 Kexin. 3.4.21.62 Subtilisin. 3.4.21.63 Oryzin.3.4.21.64 Proteinase K. 3.4.21.65 Thermomycolin. 3.4.21.66 Thermitase.3.4.21.67 Endopeptidase So. 3.4.21.68 T-plasminogen activator. 3.4.21.69Protein C (activated). 3.4.21.70 Pancreatic endopeptidase E. 3.4.21.71Pancreatic elastase II. 3.4.21.72 IgA-specific serine endopeptidase.3.4.21.73 U-plasminogen activator. 3.4.21.74 Venombin A. 3.4.21.75Furin. 3.4.21.76 Myeloblastin. 3.4.21.77 Semenogelase. 3.4.21.78Granzyme A. 3.4.21.79 Granzyme B. 3.4.21.80 Streptogrisin A. 3.4.21.81Streptogrisin B. 3.4.21.82 Glutamyl endopeptidase II. 3.4.21.83Oligopeptidase B. 3.4.21.84 Limulus clotting factor C. 3.4.21.85 Limulusclotting factor B. 3.4.21.86 Limulus clotting enzyme. 3.4.21.87 Omptin.3.4.21.88 Repressor lexA. 3.4.21.89 Signal peptidase I. 3.4.21.90Togavirin. 3.4.21.91 Flavirin. 3.4.21.92 Endopeptidase Clp. 3.4.21.93Proprotein convertase 1. 3.4.21.94 Proprotein convertase 2. 3.4.21.95Snake venom factor V activator. 3.4.21.96 Lactocepin. 3.4.22.1 CathepsinB. 3.4.22.2 Papain. 3.4.22.3 Ficain. 3.4.22.6 Chymopapain. 3.4.22.7Asclepain. 3.4.22.8 Clostripain. 3.4.22.10 Streptopain. 3.4.22.14Actinidain. 3.4.22.15 Cathepsin L. 3.4.22.16 Cathepsin H. 3.4.22.17Calpain. 3.4.22.24 Cathepsin T. 3.4.22.25 Glycyl endopeptidase.3.4.22.26 Cancer procoagulant. 3.4.22.27 Cathepsin S. 3.4.22.28Picornain 3C. 3.4.22.29 Picornain 2A. 3.4.22.30 Caricain. 3.4.22.31Ananain. 3.4.22.32 Stem bromelain. 3.4.22.33 Fruit bromelain. 3.4.22.34Legumain. 3.4.22.35 Histolysain. 3.4.22.36 Caspase-1. 3.4.22.37Gingipain R. 3.4.22.38 Cathepsin K. 3.4.23.1 Pepsin A. 3.4.23.2 PepsinB. 3.4.23.3 Gastricsin. 3.4.23.4 Chymosin. 3.4.23.5 Cathepsin D.3.4.23.12 Neopenthesin. 3.4.23.15 Renin. 3.4.23.16 Retropepsin.3.4.23.17 Pro-opiomelanocortin converting enzyme. 3.4.23.18Aspergillopepsin I. 3.4.23.19 Aspergillopepsin II. 3.4.23.20Penicillopepsin. 3.4.23.21 Rhizopuspepsin. 3.4.23.22 Endothiapepsin.3.4.23.23 Mucoropepsin. 3.4.23.24 Candidapepsin. 3.4.23.25Saccharopepsin. 3.4.23.26 Rhodotorulapepsin. 3.4.23.27 Physaropepsin.3.4.23.28 Acrocylindropepsin. 3.4.23.29 Polyporopepsin. 3.4.23.30Pycnoporopepsin. 3.4.23.31 Scytalidopepsin A. 3.4.23.32 ScytalidopepsinB. 3.4.23.33 Xanthomonapepsin. 3.4.23.34 Cathepsin E. 3.4.23.35Barrierpepsin. 3.4.23.36 Signal peptidase II. 3.4.23.37Pseudomonapepsin. 3.4.23.38 Plasmepsin I. 3.4.23.39 Plasmepsin II.3.4.23.40 Phytepsin. 3.4.24.1 Atrolysin A. 3.4.24.3 Microbialcollagenase. 3.4.24.6 Leucolysin. 3.4.24.7 Interstitial collagenase.3.4.24.11 Neprilysin. 3.4.24.12 Envelysin. 3.4.24.13 IgA-specificmetalloendopeptidase. 3.4.24.14 Procollagen N-endopeptidase. 3.4.24.15Thimet oligopeptidase. 3.4.24.16 Neurolysin. 3.4.24.17 Stromelysin 1.3.4.24.18 Meprin A. 3.4.24.19 Procollagen C-endopeptidase. 3.4.24.20Peptidyl-Lys metalloendopeptidase. 3.4.24.21 Astacin. 3.4.24.22Stromelysin 2. 3.4.24.23 Matrilysin. 3.4.24.24 Gelatinase A. 3.4.24.25Aeromonolysin. 3.4.24.26 Pseudolysin. 3.4.24.27 Thermolysin. 3.4.24.28Bacillolysin. 3.4.24.29 Aureolysin. 3.4.24.30 Coccolysin. 3.4.24.31Mycolysin. 3.4.24.32 Beta-lytic metalloendopeptidase. 3.4.24.33Peptidyl-Asp metalloendopeptidase. 3.4.24.34 Neutrophil collagenase.3.4.24.35 Gelatinase B. 3.4.24.36 Leishmanolysin. 3.4.24.37Saccharolysin. 3.4.24.38 Autolysin. 3.4.24.39 Deuterolysin. 3.4.24.40Serralysin. 3.4.24.41 Atrolysin B. 3.4.24.42 Atrolysin C. 3.4.24.43Atroxase. 3.4.24.44 Atrolysin E. 3.4.24.45 Atrolysin F. 3.4.24.46Adamalysin. 3.4.24.47 Horrilysin. 3.4.24.48 Ruberlysin. 3.4.24.49Bothropasin. 3.4.24.50 Bothrolysin. 3.4.24.51 Ophiolysin. 3.4.24.52Trimerelysin I. 3.4.24.53 Trimerelysin II. 3.4.24.54 Mucrolysin.3.4.24.55 Pitrilysin. 3.4.24.56 Insulysin. 3.4.24.57 O-sialoglycoproteinendopeptidase. 3.4.24.58 Russellysin. 3.4.24.59 Mitochondrialintermediate peptidase. 3.4.24.60 Dactylysin. 3.4.24.61 Nardilysin.3.4.24.62 Magnolysin. 3.4.24.63 Meprin B. 3.4.24.64 Mitochondrialprocessing peptidase. 3.4.24.65 Macrophage elastase. 3.4.24.66Choriolysin L. 3.4.24.67 Choriolysin H. 3.4.24.68 Tentoxilysin.3.4.24.69 Bontoxilysin. 3.4.24.70 Oligopeptidase A. 3.4.24.71Endothelin-converting enzyme 1. 3.4.24.72 Fibrolase. 3.4.24.73Jararhagin. 3.4.24.74 Fragilysin. 3.4.99.46 Multicatalytic endopeptidasecomplex.

[0321] In certain preferred embodiments, proteases that are useful ascatalytic moieties in the present invention include: serine proteasessuch as chymotrypsin, trypsin, elastase, plasmin, tissue-typeplasminogen activator (t-PA), urokinase (UK), single-chain urokinase(scu-PA), thrombin, kallikrein, acrosin, cathepsin G, coagulationfactors VIIa, IXa and XIa; cysteine proteases such as cathepsin B,papain, ficin, chymopapain, clostripain and cathepsin L; and acidproteases such as the pepsins, chymosin and cathepsin D.

[0322] Many purified serine proteases are commercially available,including: leukocyte elastase from human leukocytes (Sigma Catalog No.E1508; pancreatic elastase from human sputum (Sigma Catalog No. E1633);plasmin from human plasma (Sigma Catalog No. P4895); single-chain t-PAfrom human melanoma cell cultures (Sigma Catalog No. T7776); recombinanttwo-chain t-PA (Sigma Catalog No. T4654); urokinase from human kidneycells (Sigma Catalog No. U5004); urokinase from human urine (SigmaCatalog No. U6876); Trypsin (Sigma Catalog No. T8003); andalpha-Chymotrypsin (Sigma Catalog No. C7762).

[0323] Other useful enzymes include: pancreatic lipase; lipoproteinlipases; monoglyceride lipase; sphingosyl-glucopyranoside;sphingomyelinase; phosphoinosisides; phospholipases; peptidases such ascarboxypeptidases, aminopeptidases and dipeptidases; glucosidases;glucanases; galactosidases; mannosidases; amylases and dextrinases.

[0324] The catalytic domain of the adzymes can also be derived from anengineered enzyme, e.g., a protein engineered to have an enzymaticactivity, such as a protein designed to contain a serine protease activemotif (see, for example, Quemenur et al. (1998) Nature 391:301-304 andLiu et al. (1998) Molecular Immunology 15:1069-1077).

[0325] As a further example, the catalytic moiety can be a catalyticantibody. Because antibodies can be generated that selectively bindalmost any molecule of interest, this technology offers the potential totailor-make highly selective catalysts. Methods for making catalyticantibodies are disclosed by Lemer et al. (1991) Science 252:659;Benkovic et al. (1990) Science 250:1135; Tramontano et al. (1986)Science 234:1566. Alternatively, tailoring of an antibody to create acatalytic antibody can be carried out by methods such as walk-throughmutagenesis (see PCT application PCT/US91/02362, incorporated byreference herein).

[0326] C. Generating Chimeric Adzymes

[0327] The catalytic moiety can be linked to the targeting moiety in anumber of ways including by cotranslation from a recombinant nucleicacid (e.g., fusion proteins) or, in less preferred embodiments, chemicalcoupling.

[0328] (i) Generated as Recombinant Fusion Proteins

[0329] The adzymes of this invention can be constructed as a fusionprotein, containing the catalytic moiety and the targeting moiety as onecontiguous polypeptide chain. In preparing the fusion protein, a fusiongene is constructed comprising DNA encoding the sequences for thetargeting moiety, the catalytic moiety, and optionally, a peptide linkersequence to span the two fragments. To make this fusion protein, anentire enzyme can be cloned and expressed as part of the protein, oralternatively, a suitable fragment containing the catalytic moiety canbe used. Likewise, the entire cloned coding sequence of a targetingmoiety such as a receptor or antibody, or alternatively, a fragment ofthe molecule capable of binding the surface component of the pathogencan be used. The use of recombinant DNA techniques to create a fusiongene, with the translational product being the desired fusion protein,is well known in the art. Both the coding sequence of a gene and itsregulatory regions can be redesigned to change the functional propertiesof the protein product, the amount of protein made, or the cell type inwhich the protein is produced. The coding sequence of a gene can beextensively altered—for example, by fusing part of it to the codingsequence of a different gene to produce a novel hybrid gene that encodesa fusion protein. Examples of methods for producing fusion proteins aredescribed in PCT applications PCT/US87/02968, PCT[US89/03587 andPCT/US90/07335, as well as Traunecker et al. (1989) Nature 339:68.

[0330] Signal peptides facilitate secretion of proteins from cells. Anexemplary signal peptide is the amino terminal 25 amino acids of theleader sequence of murine interleukin7 (IL-7; Namen et al., Nature333:571; 1988). Other signal peptides may also be employed furthermore,certain nucleotides in the IL-7 leader sequence can be altered withoutaltering the amino acid sequence. Additionally, amino acid changes thatdo not affect the ability of the IL-7 sequence to act as a leadersequence can be made. A signal peptide may be added to the fusion adzymetarget domain or catalytic domain, such that when these domains aresynthesized by cells from transfected nucleic acids, the secreted adzymetarget and catalytic domains will oligomerize to form mature adzymes toact on extracellular targets, such as cytokines.

[0331] In some instances it may be necessary to introduce a polypeptidelinker region between portions of the chimeric protein derived fromdifferent proteins. This linker can facilitate enhanced flexibility ofthe fusion protein allowing various portions to freely and (optionally)simultaneously interact with a target by reducing steric hindrancebetween the portions, as well as allowing appropriate folding of eachportion to occur. The linker can be of natural origin, such as asequence determined to exist in random coil between two domains of aprotein. Alternatively, the linker can be of synthetic origin. Forinstance, the sequence (Gly₄Ser)_(n) can be used as a syntheticunstructured linker. Linkers of this type are described in Huston et al.(1988) PNAS 85:4879; and U.S. Pat. Nos. 5,091,513 and 5,258,498.Naturally occurring unstructured linkers of human origin are preferredas they reduce the risk of immunogenicity.

[0332] The length and composition of the linker connecting the addressand the catalytic domain may be optimized. For example, Zhou (J. Mol.Biol. 329: 1-8, 2003) describes in detail a quantitative theory forenhancing affinity for a first molecule by linking a second and a thirdmolecule (such as two scFvs), each of which has affinity for the firstmolecule. The predicted affinity enhancement is found to be actuallyapproached by a bi-specific antibody against hen egg lysozyme consistingof scFv fragments of D1.3 and HyHEL-10. The wide applicability of thetheory is demonstrated by diverse examples of protein-proteininteractions constrained by flexible linkers, and the theory provides ageneral framework for understanding protein-protein interactionsconstrained by flexible linkers.

[0333] In the simplest case of the theory, the linker is flexible suchthat its only effect is to provide a leash constraining the distancesbetween the two antibody fragments. Then it was shown:

C _(eff) =p(d ₀)  (Eq. a)

[0334] where p(r) is the probability density for the end-to-end vectorof the flexible linker with L residues to have a distance r, and d₀ isthe actual end-to-end distance when the linked fragments are bound tothe antigen. A flexible peptide linker consisting of L residues can bemodeled as a worm-like chain, such that:

p(r)=(3/4l _(p) l _(c))^(3/2) exp(−3r ²/4l _(p) l _(c))(1−5l _(p)/4l_(c)+2r ² /l ^(c) ²−33r ⁴/80l _(p) l _(c) ³−79l _(p) ²/160l _(c) ³−329r₂ l _(p)/120l _(c) ³+6799r ⁴/1600l _(c) ⁴−344^(lr) ⁶/2800l _(p) l _(c)⁵+1089r ⁸/12800l _(p) ² l _(c) ⁶)  (Eq. b)

[0335] where b=3.8 Å is the nearest C_(α)-C_(α) distance, and l_(c)=bLand l_(p)=3 Å are the contour length and persistence length,respectively, of the peptide linker. Typically p(d₀) is in themillimolar range or higher, and hence the linking strategy is expectedto result in significant affinity enhancement, since the associationconstants of antibody fragments are much greater than 10³ M-⁻¹. Equation(a) has been found to predict well the affinity enhancements of linkingDNA-binding domains (Zhou, Biochemistry 40, pp. 15069-15073, 2001).

[0336] Based on this theoritic model, FIG. 2 of Zhou (incorporated byreference) describes the relationship of L and p(d₀) at several given d₀values, such as 10 Å, 20 Å, 30 Å, 40 Å, 50 Å, and 60 Å. This linkertheory incorporates two important realistic aspects. First, in the boundstate, the end-to-end distance of the linker is kept at around aspecific value (d₀) determined by the structure of the bound complex.Second, in the unbound state, the distribution p(r) of the end-to-enddistance is not uniform but is what is appropriate for a semi-flexiblepolymer chain, such as a polypeptide chain. For entropic reasons, apolymer chain very rarely samples conformations with end-to-enddistances approaching either zero or the full contour length l_(c), thusp(r) has a maximum at an intermediate value of r. At a given end-to-enddistance d₀, there is also a value of l_(c) (or L) at which p(d₀) ismaximal (see FIG. 2 of Zhou). Therefore, the chain length of a peptidelinker can be optimized to achieve maximal affinity enhancement.

[0337] In the context of the adzyme linker design, once the address andthe catalytic domain is chosen, molecular model of the target—adzymeavid complex may be obtained. d₀, the distance between the point wherethe linker connects to the address and the point where the linkerconnects to the enzyme, while both the address and enzyme domain are inthe avid complex, can be readily determined from, for example, the 3-Dstructure of the target—adzyme complex. Many cytokine structures aresolved (see the Cytokine Web site athttp:/cmbi.bjmu.edu.cn/cmbidata/cgf/CGF_Database/cytweb/cyt_strucs/index.html).The structure of those other cytokines with sequence homology tocytokines of known structures, as well as the target—adzyme complex maybe routinely obtained via molecular modeling.

[0338] Once the d₀ value is obtained, FIG. 2 of Zhou may be used to findthe optimum L for the highest possible p(d₀) value. For example, if itis determined that d₀ is about 20 Å, FIG. 2 of Zhou indicates that atthis d₀ value, the highest possible p(d₀) value is about 20 mM, and thatp(d₀) value corresponds to a linker length of about 10-15 amino acids.Note that at d₀ value larger than 20 Å, the maximum p(d₀) value peaksquickly and tapers off very gradually, thus allowing quite a bit offlexibility in chosing a proper linker length. In addition, the methodhere is rather tolerant of a relatively imprecise estimation of the d₀value, since in FIG. 2 of Zhou, curves for different u₀ values tend toconverge, especially in long linker length (e.g., more than 40 aminoacids) and large d₀ values (30-60). For example, when d₀ is 30, the peakp(d₀) value is about 3-4 mM. When d₀ is 40, the peak p(d₀) onlydecreases to about 1.5 mM, at about the same linker length of around35-40 residues.

[0339] Techniques for making fusion genes are well known. Essentially,the joining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a fusion gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992).

[0340] Fusion proteins can comprise additional sequences, including aleader (or signal peptide) sequence, a portion of an immunoglobulin(e.g., an Fc portion, see below) or other oligomer-forming sequences, aswell as sequences encoding highly antigenic moieties, hexahistidinemoieties or other elements that provide a means for facile purificationor rapid detection of a fusion protein.

[0341] To express the fusion protein molecule, it may be desirable toinclude transcriptional and translational regulatory elements and othernon-coding sequences to the fusion gene construct. For instance,regulatory elements including constituitive and inducible promoters,enhancers or inhibitors can be incorporated.

[0342] (ii) Use of Chemical Coupling Agents

[0343] There are a large number of chemical cross-linking agents thatare known to those skilled in the art. For the present invention, thepreferred cross-linking agents are heterobifunctional cross-linkers,which can be used to link proteins in a stepwise manner.Heterobifunctional cross-linkers provide the ability to design morespecific coupling methods for conjugating proteins, thereby reducing theoccurrences of unwanted side reactions such as homo-protein polymers. Awide variety of heterobifunctional crosslinkers are known in the art.These include: succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl(4-iodoacetyl) aminobenzoate (SLAB), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC);4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-tolune (SMPT),N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl6-[(3-(2-pyridyldithio)propionate]hexanoate (LC-SPDP). Thosecross-linking a gents having N-hydroxysuccinimide moieties can beobtained as the N-hydroxysulfosuccinimide analogs, which generally havegreater water solubility. In addition, those cross-linking agents havingdisulfide bridges within the linking chain can be synthesized instead asthe alkyl derivatives so as to reduce the amount of linker cleavage invivo.

[0344] In addition to the heterobifunctional cross-linkers, there existsa number of other cross-linking agents including homobifunctional andphotoreactive cross-linkers. Disuccinimidyl suberate (DSS),bismaleimidohexane (BMH) and dimethylpimelimidate-2 HCl (DMP) areexamples of usefull homobifunctional cross-linking agents, andbis[.beta.-(4-azidosalicylamido)ethyl]disulfide (BASED) andN-succinimidyl-6(4′-azido-2′-nitrophenyl-amino)hexanoate (SANPAH) areexamples of useful photoreactive cross-linkers for use in thisinvention. For a review of protein coupling techniques, see Means et al.(1990) Bioconjugate Chemistry 1:2-12. One particularly useful class ofheterobifunctional cross-linkers, included above, contain the primaryamine reactive group, N-hydroxysuccinimide (NHS), or its water solubleanalog N-hydroxysulfosuccinimide (sulfo-NHS). Primary amines (lysineepsilon groups) at alkaline pH's are unprotonated and react bynucleophilic attack on NHS or sulfo-NHS esters. This reaction results inthe formation of an amide bond, and release of NHS or sulfo-NHS as aby-product.

[0345] Another reactive group useful as part of a heterobifunctionalcross-linker is a thiol reactive group. Common thiol reactive groupsinclude maleimides, halogens, and pyridyl disulfides. Maleimides reactspecifically with free sulfhydryls (cysteine residues) in minutes, underslightly acidic to neutral (pH 6.5-7.5) conditions. Halogens (iodoacetylfunctions) react with --SH groups at physiological pH's. Both of thesereactive groups result in the formation of stable thioether bonds.

[0346] The third component of the heterobifunctional cross-linker is thespacer arm or bridge. The bridge is the structure that connects the tworeactive ends. The most apparent attribute of the bridge is its effecton steric hindrance. In some instances, a longer bridge can more easilyspan the distance necessary to link two complex biomolecules.

[0347] Preparing protein-protein conjugates using heterobifunctionalreagents is a two-step process involving the amine reaction and thesulfhydryl reaction, and such processes are, in view of thisspecification, generally well known in the art. See, e.g., Partis et al.(1983) J. Pro. Chem. 2:263); Ellman et al. (1958) Arch. Biochem.Biophys. 74:443; Riddles et al. (1979) Anal. Biochem. 94:75); Blattleret al. (1985) Biochem 24:1517).

[0348] (iii) Multimeric Constructs

[0349] In certain embodiments of the invention, the subject adzyme is amultimeric complex in which the catalytic domain and targeting domainare on separate polypeptide chains. These two domains, when synthesized,can be brought together to form the mature adzyme.

[0350] For example, in one embodiment, the adzyme takes the form of anantibody (e.g., Fc fusion) in which the variable regions of the heavy(V_(H)) and light chain (V_(L)) have been replaced with the targetingand catalytic domains (either the targeting or the catalytic domain canreplace either the V_(H) region or the V_(L) region). For example,soluble proteins comprising an extracellular domain from amembrane-bound protein and an immunoglobulin heavy chain constant regionwas described by Fanslow et al., J. Immunol. 149:65, 1992 and by Noelleet al., Proc. Nad. Acad. Sci. U.S.A. 89:6550, 1992.

[0351] In certain embodiments, an adzyme comprises a first Fc portionthat is connected to the appropriate heavy and light chains which mayfunction as a targeting moiety, and a second Fc portion that is fused toa catalytic domain.

[0352] Fusion proteins comprising a catalytic domain or a targetingdomain may be prepared using nucleic acids encoding polypeptides derivedfrom immunoglobulins. Preparation of fusion proteins comprisingheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., (PNAS USA 88:10535, 1991) and Byrn et al., (Nature344:677, 1990). In one embodiment of the invention, an adzyme is createdby fusing a catalytic domain to a first Fc region of an antibody (e.g.,IgG1) and a targeting domain to a second Fc region of an antibody. TheFc polypeptide preferably is fused to the C-terminus of a catalytic ortargeting domain. A gene fusion encoding each Fc fusion protein isinserted into an appropriate expression vector. The Fc fusion proteinsare expressed in host cells transformed with the recombinant expressionvector, and allowed to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between Fc polypeptides, yielding thedesired adzymes. If fusion proteins are made with both heavy and lightchains of an antibody, it is possible to form an adzyme with multiplecatalytic and targeting domains.

[0353] In certain embodiments, an adzyme comprising one or moreimmunoglobulin fusion protein may employ an immunoglobulin light chainconstant region in association with at least one immunoglobulin heavychain constant region domain. In another embodiment, an immunoglobulinlight chain constant region is associated with at least oneimmunoglobulin heavy chain constant region domain joined to animmunoglobulin hinge region. In one set of embodiments, animmunoglobulin light chain constant region joined in frame with apolypeptide chain of a non-immunoglobulin polypeptide (e.g., a catalyticdomain or polypeptide targeting domain) and is associated with at leastone heavy chain constant region. In a preferred set of embodiments avariable region is joined upstream of and in proper reading frame withat least one immunoglobulin heavy chain constant region. In another setof embodiments, an immunoglobulin heavy chain is joined in frame with apolypeptide chain of a non-immunoglobulin polypeptide and is associatedwith an immunoglobulin light chain constant region. In yet another setof embodiments, a polypeptide chain of a non-immunoglobulin polypeptidedimer or receptor analog is joined to at least one immunoglobulin heavychain constant region which is joined to an immunoglobulin hinge regionand is associated with an immunoglobulin light chain constant region. Ina preferred set of embodiments an immunoglobulin variable region isjoined upstream of and in proper reading frame with the immunoglobulinlight chain constant region.

[0354] The term “Fc polypeptide” as used herein includes native andaltered forms of polypeptides derived from the Fc region of an antibody.Truncated froms of such polypeptides containing the hinge region thatpromotes dimerization are also included. One suitable Fc polypeptide,described in PCT application WO 93/10151, is a single chain polypeptideextending from the N-terminal hinge region to the native C-terminus. Itmay be desirable to use altered forms of Fc polypeptides having improvedserum half-life, altered spatial orientation, and the like.Immunoglobulin heavy chain constant region domains include C_(H)1,C_(H)2, C_(H)3, and C_(H)4 of any class of immunoglobulin heavy chainincluding gamma, alpha, epsilon, mu, and delta classes. A particularlypreferred immunoglobulin heavy chain constant region domain is humanC_(H)1. Immunoglobulin variable regions include V_(H), V_(kappa), orV_(tambda) DNA sequences encoding immunoglobulins may be cloned from avariety of genomic or cDNA libraries known in the art. The techniquesfor isolating such DNA sequences using probe-based methods areconventional techniques and are well known to those skilled in the art.Probes for isolating such DNA sequences may be based on published DNAsequences (see, for example, Hieter et al., Cell 22: 197-207, 1980).Alternatively, the polymerase chain reaction (PCR) method disclosed byMullis et al. (U.S. Pat. No. 4,683,195) and Mullis (U.S. Pat. No.4,683,202), incorporated herein by reference may be used. The choice oflibrary and selection of probes for the isolation of such DNA sequencesis within the level of ordinary skill in the art.

[0355] Host cells for use in preparing immunoglobulin fusions includeeukaryotic cells capable of being transformed or transfected withexogenous DNA and grown in culture, such as cultured mammalian andfungal cells. Fungal cells, including species of yeast (e.g.,Saccharomvces spp., Schizosaccharomyces spp.), or filamentous fungi(e.g., Aspergillus spp., Neurospora spp.) may be used as host cellswithin the present invention. Strains of the yeast Saccharomycescerevisiae are particularly preferred.

[0356] In each of the foregoing embodiments, a molecular linkeroptionally may be interposed between, and covalently join, the rest ofthe adzyme construct and the dimerization domain.

[0357] In another embodiment, various oligomerization domains may beemployed to bring together the separately synthesized targeting andcatalytic domains.

[0358] One class of such oligomerization domain is leucine zipper. WO94/10308 A1 and its related U.S. Pat. No. 5,716,805 (all incorporatedherein by reference) describes the use of leucine zipper oligomerizationdomains to dimerize/oligomerize two separate heterologous polypeptides.Each of the two separate heterologous polypeptides is synthesized as afusion protein with a leucine zipper oligomerization domain. In oneembodiment, the leucine zipper domain can be removed from the fusionprotein, by cleavage with a specific proteolytic enzyme. In anotherembodiment, a hetero-oligomeric protein is prepared by utilizing leucinezipper domains that preferentially form hetero-oligomers.

[0359] Leucine zipper domains were originally identified in severalDNA-binding proteins (Landschulz et al., Science 240:1759, 1988).Leucine zipper domain is a term used to refer to a conserved peptidedomain present in these (and other) proteins, which is responsible fordimerization of the proteins. The leucine zipper domain (also referredto herein as an oligomerizing, or oligomer-forming, domain) comprises arepetitive heptad repeat, with four or five leucine residuesinterspersed with other amino acids.

[0360] Examples of leucine zipper domains are those found in the yeasttranscription factor GCN4 and a heat-stable DNA-binding protein found inrat liver (C/EBP; Landschulz et al., Science 243:1681, 1989). Twonuclear transforming proteins, fos and jun, also exhibit leucine zipperdomains, as does the gene product of the murine proto-oncogene, c-myc(Landschulz et al., Science 240:1759, 1988). The products of the nuclearoncogenes fos and jun comprise leucine zipper domains preferentiallyform a heterodimer (O'Shea et al., Science 245:646, 1989; Turner andTjian, Science 243:1689, 1989). The leucine zipper domain is necessaryfor biological activity (DNA binding) in these proteins.

[0361] The fusogenic proteins of several different viruses, includingparamyxovirus, coronavirus, measles virus and many retroviruses, alsopossess leucine zipper domains (Buckland and Wild, Nature 338:547,1989;Britton, Nature 353:394, 1991; Delwart and Mosialos, AIDS Research andHuman Retroviruses 6:703, 1990). The leucine zipper domains in thesefusogenic viral proteins are near the transmembrane region of theproteins; it has been suggested that the leucine zipper domains couldcontribute to the oligomeric structure of the fusogenic proteins.Oligomerization of fusogenic viral proteins is involved in fusion poreformation (Spruce et al, PNAS 88:3523, 1991). Leucine zipper domainshave also been recently report ed to play a role in oligomerization ofheat-shock transcription factors (Rabindran et al., Science 259:230,1993).

[0362] Accordingly, in certain embodiments, the dimerization domains ofthe adzyme components comprise coiled-coil dimerization domains, such asleucine zipper domains. Preferably, the leucine zipper domains includeat least four leucine heptads. In one preferred embodiment, the leucinezipper domain is a Fos or Jun leucine zipper domain.

[0363] Many other so-called “bundling domains” exist which performessentially the same function of the above-described leucine zipperdomains to bring together the catalytic and target domains. For example,WO 99/10510 A2 (incorporated herein by reference) describes bundlingdomains include any domain that induces proteins that contain it to formmultimers (“bundles”) through protein-protein interactions with eachother or with other proteins containing the bundling domain. Examples ofthese bundling domains include domains such as the lac repressortetramerization domain, the p53 tetramerization domain, the leucinezipper domain, and domains derived therefrom which retain observablebundling activity. Proteins containing a bundling domain are capable ofcomplexing with one another to form a bundle of the individual proteinmolecules. Such bundling is “constitutive” in the sense that it does notrequire the presence of a crosslinking agent (i.e., a cross-linkingagent which doesn't itself contain a pertinacious bundling domain) tolink the protein molecules.

[0364] As described above, bundling domains interact with like domainsvia protein-protein interactions to induce formation of protein“bundles.” Various order oligomers (dimers, trimers, tertramers, etc.)of proteins containing a bundling domain can be formed, depending on thechoice of bundling domain.

[0365] In one embodiment, incorporation of a tetranerization domainwithin a fusion protein leads to the constitutive assembly of tetramericclusters or bundles. The E. coli lactose repressor tetramerizationdomain (amino acids 46-360; Chakerian et al. (1991) J. Biol. Chem.266.1371; Alberti et al. (1993) EMBO J. 12:3227; and Lewis et al. (1996)Nature 271:1247), illustrates this class. Other illustrativetetramerization domains include those derived from residues 322-355 ofp53 (Wang et al. (1994) Mol. Cell. Biol. 14:5182; Clore et al. (I 994)Science 265:386) see also U.S. Pat. No. 5,573,925 by Halazonetis.

[0366] In yet another embodiment, the catalytic domain and the targetdomain may each be fused to a “ligand binding domain,” which, uponbinding to a small molecule, will bring the catalytic domain and thetarget domain together (“small molecule-mediated oligomerization”).

[0367] Fusion proteins containing a ligand binding domain for use inpracticing this invention can function through one of a variety ofmolecular mechanisms.

[0368] In certain embodiments, the ligand binding domain permitsligand-mediated crosslinking of the fusion protein molecules bearingappropriate ligand binding domains. In these cases, the ligand is atleast divalent and functions as a dimerizing agent by binding to the twofusion proteins and forming a cross-linked heterodimeric complex whichactivates target gene expression. See e.g. WO 94/18317, WO 96/20951, WO96/06097, WO 97/31898 and WO 96/41865.

[0369] In the cross-linking-based dimerization systems the fusionproteins can contain one or more ligand binding domains (in some casescontaining two, three, four, or more of such domains) and can furthercontain one or more additional domains, heterologous with respect to theligand binding domain, including e.g. a catalytic or target domain ofthe subject adzyme.

[0370] In general, any ligand/ligand binding domain pair may be used insuch systems. For example, ligand binding domains may be derived from animmunophilin such as an FKBP, cyclophilin, FRB domain, hormone receptorprotein, antibody, etc., so long as a ligand is known or can beidentified for the ligand binding domain.

[0371] For the most part, the receptor domains will be at least about 50amino acids, and fewer than about 350 amino acids, usually fewer than200 amino acids, either as the natural domain or truncated activeportion thereof. Preferably the binding domain will be small (<25 kDa,to allow efficient transfection in Viral vectors), monomeric,nonimmunogenic, and should have synthetically accessible, cell permeant,nontoxic ligands as described above.

[0372] Preferably the ligand binding domain is for (i.e., binds to) aligand which is not itself a gene product (i.e., is not a protein), hasa molecular weight of less than about 5 kD and preferably less thanabout 2.5 kD, and optionally is cell permeant. In many cases it will bepreferred that the ligand does not have an intrinsic pharmacologicactivity or toxicity which interferes with its use as an oligomerizationregulator.

[0373] The DNA sequence encoding the ligand binding domain can besubjected to mutagenesis for a variety of reasons. The mutagenizedligand binding domain can provide for higher binding affinity, allow fordiscrimination by a ligand between the mutant and naturally occurringforms of the ligand binding domain, provide opportunities to designligand-ligand binding domain pairs, or the like. The change in theligand binding domain can involve directed changes in amino acids knownto be involved in ligand binding or with ligand-dependent conformationalchanges. Alternatively, one may employ random mutagenesis usingcombinatorial techniques. In either event, the mutant ligand bindingdomain can be expressed in an appropriate prokaryotic or eukaryotic hostand then screened for desired ligand binding or conformationalproperties. Examples involving FKBP, cyclophilin and FRB domains aredisclosed in detail in WO 94/18317, WO 96/06097, WO 97/31898 and WO96/41865. For instance, one can change Phe36 to Ala and/or Asp37 to Glyor Ala in FKBP12 to accommodate a substituent at positions 9 or 10 ofthe ligand FK506 or FK520 or analogs, mimics, dimers or otherderivatives thereof. In particular, mutant FKBP12 domains which containVal, Ala, Gly, Met or other small amino acids in place of one or more ofTyr26, Phe36, Asp37, Tyr82 and Phe99 are of particular interest asreceptor domains for FK506-type and FK type ligands containingmodifications at C9 and/or C10 and their synthetic counterparts (see,e.g., WO97/31898). Illustrative mutations of current interest in FKBPdomains also include the following: TABLE III Entries identify thenative amino acid by single letter code and sequence position, followedby the replacement amino acid in the mutant. Thus, F36V designates ahuman FKBP12 sequence in which phenylalanine at position 36 is replacedby valine. F36V/F99A indicates a double mutation in which phenylalanineat positions 36 and 99 are replaced by valine and alanine, respectively.F36A Y26V F46A W59A F36V Y26S F48H H87W F36M D37A F48L H87R F36S I90AF48A F36V/F99A F99A I91A E54A/F36V/F99G F99G F46H E54K/F36M/F99A Y26AF46L V55A F36M/F99G

[0374] Illustrative examples of domains which bind to the FKBP:rapamycincomplex (“FRBs”) are those which include an approximately 89-amino acidsequence containing residues 2025-2113 of human FRAP. AnotherFRAP-derived sequence of interest comprises a 93 amino acid sequenceconsisting of amino acids 2024-2113. Similar considerations apply to thegeneration of mutant FRAP-derived domains which bind preferentially toFKBP complexes with rapamycin analogs (rapalogs) containingmodifications (i.e., are ‘bumped’) relative to rapamycin in theFRAP-binding portion of the drug. For example, one may obtainpreferential binding using rapalogs bearing substituents; other than—OMe at the C7 position with FRBs based on the human FRAP FRB peptidesequence but bearing amino acid substitutions for one of more of theresidues Tyr2038, Phe2039, Thr2098, Gln2099, Trp2101 and Asp2102.Exemplary mutations include Y2038H, Y2038L, Y2038V, Y2038A, F2039H,F2039L, F2039A, F2039V, D2102A, T2098A, T2098N, T2098L, and T2098S.Rapalogs bearing substituents; other than —OH at C28 and/or substituentsother than ═O at C30 may be used to obtain preferential binding to FRAPproteins bearing an amino acid substitution for Glu2032. Exemplarymutations include E2032A and E2032S. Proteins comprising an FRBcontaining one or more amino acid replacements at the foregoingpositions, libraries of proteins or peptides randomized at thosepositions (i.e., containing various substituted amino acids at thoseresidues), libraries randomizing the entire protein domain, orcombinations of these sets of mutants are made using the proceduresdescribed above to identify mutant FRAPs that bind preferentially tobumped rapalogs.

[0375] Other macrolide binding domains useful in the present invention,including mutants thereof, are described in the art. See, for example,WO96/41865, WO96/136131 WO96/061111 WO96/061101 WO96/060971 WO96/127961WO95/053891 WO95/026842.

[0376] The ability to employ in vitro mutagenesis or combinatorialmodifications of sequences encoding proteins allows for the productionof libraries of proteins which can be screened for binding affinity fordifferent ligands. For example, one can randomize a sequence of 1 to 5,5 to 10, or 10 or more codons, at one or more sites in a DNA sequenceencoding a binding protein, make an expression construct and introducethe expression construct into a unicellular microorganism, and develop alibrary of modified sequences. One can then screen the library forbinding affinity of the encoded polypeptides to one or more ligands. Thebest affinity sequences which are compatible with the cells into whichthey would be introduced can then be used as the ligand binding domainfor a given ligand. The ligand may be evaluated with the desired hostcells to determine the level of binding of the ligand to endogenousproteins. A binding profile may be determined for each such ligand whichcompares ligand binding affinity for the modified ligand binding domainto the affinity for endogenous proteins. Those ligands which have thebest binding profile could then be used as the ligand. Phage displaytechniques, as a non-limiting example, can be used in carrying out theforegoing.

[0377] In other embodiments, antibody subunits, e.g. heavy or lightchain, particularly fragments, more particularly all or part of thevariable region, or single chain antibodies, can be used as the ligandbinding domain. Antibodies can be prepared against haptens which arepharmaceutically acceptable and the individual antibody subunitsscreened for binding affinity. cDNA encoding the antibody subunits canbe isolated and modified by deletion of the constant region, portions ofthe variable region, mutagenesis of the variable region, or the like, toobtain a binding protein domain that has the appropriate affinity forthe ligand. In this way, almost any physiologically acceptable haptencan be employed as the ligand. Instead of antibody units, naturalreceptors can be employed, especially where the binding domain is known.In some embodiments of the invention, a fusion protein comprises morethan one ligand binding domain. For example, a DNA binding domain can belinked to 2, 3 or 4 or more ligand binding domains. The presence ofmultiple ligand binding domains means that ligand-mediated cross-linkingcan recruit multiple fusion proteins containing transcription activationdomains to the DNA binding domain-containing fusion protein.

[0378] Cross-linking/dimerization systems Any ligand for which a bindingprotein or ligand binding domain is known or can be identified may beused in combination with such a ligand binding domain in carrying outthis invention.

[0379] Extensive guidance and examples are provided in WO 94/18317 forligands and other components useful for cross-linkedoligomerization-based systems. Systems based on ligands for animmunophilin such as FKBP, a cyclophilin, and/or FRB domain are ofspecial interest. Illustrative examples of ligand binding domain/ligandpairs that may be used for cross-linking include, but are not limitedto: FKBP/FK1012, FKBP/synthetic divalent FKBP ligands (see WO 96/06097and WO 97/31898), FRB/rapamycin or analogs thereof:FKBP (see e.g., WO93/33052, WO 96/41865 and Rivera et al, “A humanized system forpharmacologic control of gene expression”, Nature Medicine2(9):1028-1032 (1997)), cyclophilin/cyclosporin (see e.g. WO 94/18317),FKBP/FKCsA/cyclophilin (see e.g. Belshaw et al, 1996, PNAS93:4604-4607), DHFR/methotrexate (see e.g. Licitra et al, 1996, Proc.Natl. Acad. Sci. USA 93:12817-12821), and DNA gyrase/coumermycin (seee.g. Farrar et al, 1996, Nature 383:178-181). Numerous variations andmodifications to ligands and ligand binding domains, as well asmethodologies for designing, selecting and/or characterizing them, whichmay be adapted to the present invention are disclosed in the citedreferences.

[0380] In addition, small molecule dimerizers, such as those describedin ARIAD Pharmaceutical's ARGENT homodimerization kit and ARGENT™heterodimerization kit may be used for this purpose. The ARGENT™Regulated Homodimerization Kit contains reagents for bringing togethertwo molecules of an engineered fusion protein by adding a small molecule“dimerizer.” The kit can be used to bring together any two proteins thatnormally do not interact with each other.

[0381] There are two classes of dimerizers. Homodimerizers incorporatetwo identical binding motifs, and can therefore be used to induceassociation of two proteins containing the same dimerizer-binding motif.Heterodimerizers incorporate two different binding motifs, one on eachof the two proteins, and can therefore be used to induce association ofthe two proteins containing these dimerizer-binding motifs. The ARGENT™Kits also provides a homodimerizer or a heterodimerizer, and DNA vectorsfor making appropriate fusion proteins.

[0382] The reagents in the ARGENT Kits are based on the human proteinFKBP12 (FKBP, for FK506 binding protein) and its small molecule ligands.FKBP is an abundant cytoplasmic protein that serves as the initialintracellular target for the natural product immunosuppressive drugsFK506 and rapamycin. In the original homodimerizer system developed bythe Schreiber and Crabtree laboratories (Science 262: 1019-24, 1993), adimerizer was created by chemically linking two molecules of FK506 in amanner that eliminated immunosuppressive activity. The resultingmolecule, called FK1012, was able to crosslink fusion proteinscontaining wild-type FKBP domains.

[0383] A second generation FKBP homodimerizer, AP1510, was subsequentlydeveloped (Amara et al., Proc Natl Acad Sci USA 94: 10618-23, 1997).AP1510 has the advantages of being completely synthetic, as well asbeing smaller and simpler than FK1012 and more potent in manyapplications. Other improved versions have also been developed (Clacksonet al., Proc Natl Acad Sci USA 95: 10437-42, 1998; AP1903, AP20187). TheAP20187-based system has the advantages of working at lowerconcentrations, and AP20187 has better pharmacokinetic properties thanAP1510, allowing it to be used in vivo. Other similar systems may alsobe used to bring two macromolecules together. For example, Lin et al.,(J. Am. Chem. Soc., 122, 4247-4248, 2000; also featured in Chem. & Eng.News, 78, 52, 2000) use Dexamethasone-Methotrexate as an efficientchemical inducer of protein dimerization in vivo.

[0384] iv. General Methodologies

[0385] In applications of the invention involving the geneticengineering of cells within (or for use within) whole animals, the useof peptide sequence derived from that species is preferred whenpossible. For instance, for applications involving human therapy, theuse of catalytic or targeting domains derived from human proteins mayminimize the risk of immunogenic reactions. For example, a single chainantibody to be used as a targeting moiety may preferably be a humanizedor human-derived single chain antibody. Likewise, other portions ofadzymes, such as Fc portions or oligomerization domains may be matchedto the species in which the adzyme is to be used.

[0386] E. Miscellaneous Features for Adzymes

[0387] (i) Serum Half-Life

[0388] In certain embodiments of the invention, the subject adzyme canbe designed or modified to exibit enhanced or decreased serum half-life.Enhanced serum half-life may be desirable to reduce the frequency ofdosing that is required to achieve therapeutic effectiveness. Forexample, the rate of reaction between an adzyme and a low-abundance(e.g., fempto- or pico-molar) substrate, such as certain extracellularsignaling molecules, may occur on a timescale of days to weeks;accordingly, a serum half-life allowing adzyme to persist in the bodyfor days or weeks would be desirable and would decrease the frequency ofdosing that is needed. Accordingly, in certain embodiments, the serumhalf-life of an adzyme is at least one day, and preferably two, three,five, ten, twenty or fifty days or more. Decreased serum half-life maybe desirable in, for example, acute situations, where swift alterationof a substrate will generally accomplish the desired therapeutic effect,with little added benefit resulting from prolonged adzyme activity. Infact, it may be possible to deliver very high levels of an adzyme with ashort half-life, such that a high level of therapeutic effectiveness israpidly achieved, but the adzyme is quickly cleared from the body so asto reduce side effects that may be associated with high dosages.Examples of acute situations include poisonings with various toxins,where the adzyme neutralizes or otherwise eliminates the toxin, as wellas sepsis or other severe fevers, where removal of endogenous pyrogens,such as IL-1 or TNF-α, or exogenous pyrogens, such as bacteriallipopolysaccharides, may accomplish the therapeutic purpose.

[0389] Serum half-life may be determined by a variety of factors,including degradation, modification to an inactive form and clearance bythe kidneys. For example, an effective approach to confer resistance topeptidases acting on the N-terminal or C-terminal residues of apolypeptide is to add chemical groups at the polypeptide termini, suchthat the modified polypeptide is no longer a substrate for thepeptidase. One such chemical modification is glycosylation of thepolypeptides at either or both termini. Certain chemical modifications,in particular polyethylene glycols (“pegylation”) and N-terminalglycosylation, have been shown to increase the half-life of polypeptidesin human serum (Molineux (2003), Pharmacotherapy 8 Pt 2:3S-8S.Powell etal. (1993), Pharma. Res. 10: 1268-1273). Other chemical modificationswhich enhance serum stability include, but are not limited to, theaddition of an N-terminal alkyl group, consisting of a lower alkyl offrom 1 to 20 carbons, such as an acetyl group, and/or the addition of aC-terminal amide or substituted amide group.

[0390] In certain embodiments, an adzyme may be modified, so as toincrease the hydrodynamic volume of the adzyme, thereby, among otherthings, reducing elimination from the kidneys. For example, modificationwith an inert polymer, such as polyethylene glycol, tends to decreaseelimination through the kidneys. A polymer may be of any effectivemolecular weight, and may be branched or unbranched. For polyethyleneglycol, the preferred molecular weight is between about 1 kDa and about100 kDa (the term “about” indicating that in preparations ofpolyethylene glycol, some molecules will weigh more, some less, than thestated molecular weight) for ease in handling and manufacturing. Othersizes may be used, depending on the desired therapeutic profile (e.g.,the duration of sustained release desired, the effects, if any onbiological activity, the ease in handling, the degree or lack ofantigenicity and other known effects of the polyethylene glycol to atherapeutic protein or analog). For example, the polyethylene glycol mayhave an average molecular weight of about 200, 1000, 5000, 15,000,30,000 50,000, or 100,000 kDa or more. The polyethylene glycol may havea branched structure. Branched polyethylene glycols are described, forexample, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem.Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646(1999). The polyethylene glycol molecules (or other chemical moieties)may be attached to the adzyme with consideration of effects on catalyticor targeting portions. There are a number of attachment methodsavailable to those skilled in the art, e.g., EP 0 401 384, hereinincorporated by reference (coupling PEG to G-CSF), see also Malik etal., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSFusing tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the Nterminus or lysine group.

[0391] Adzymes may be designed to have a molecular weight of about 50kilodaltons or greater so as to reduce elimination through the kidneys.

[0392] The presence of an N-terminal D-amino acid also increases theserum stability of a polypeptide that otherwise contains L-amino acids,because exopeptidases acting on the N-terminal residue cannot utilize aD-amino acid as a substrate. Similarly, the presence of a C-terminalD-amino acid also stabilizes a polypeptide, because serum exopeptidasesacting on the C-terminal residue cannot utilize a D-amino acid as asubstrate. With the exception of these terminal modifications, the aminoacid sequences of polypeptides with N-terminal and/or C-terminal D-aminoacids are usually identical to the sequences of the parent L-amino acidpolypeptide.

[0393] Substitution of unnatural amino acids for natural amino acids ina subsequence of a polypeptide can confer or enhance desirableattributes including biological activity. Such a substitution can, forexample, confer resistance to proteolysis by exopeptidases acting on theN-terminus. The synthesis of polypeptides with unnatural amino acids isroutine and known in the art (see, for example, Coller, et al. (1993),cited above).

[0394] In another embodiment, adzyme peptides are fused to certainpolypeptides to achieve enhanced/increased serum stability or half life.For example, WO 97/34631 A1 describes recombinant vectors encodingimmunoglobulin-like domains and portions thereof, such as antibodyFc-hinge fragments, subfragments and mutant domains with extendedbiological half lives. Such vectors can be used to generate largequantities of fusions with such domains following expression by hostcells. These antibody Fc and Fc-hinge domains have the same in vivostability as intact antibodies. The application also discloses domainsengineered to have increased in vivo half lives. These DNA constructsand protein domains can be adapted for use in the instant invention,such as for the production of recombinant adzymes (or adzyme components)with increased stability and longevity for therapeutic and diagnosticuses.

[0395] Specifically, WO 97/34631 A1 describes recombinant vectorsencoding immunoglobulin-like domains and portions thereof, such asantibody Fc fragments and subfragments and Fc-hinge domains withextended in vivo half lives. As the invention is exemplified by theproduction of a variety of immunoglobulin-like domains, includingantibody Fc-hinge, Fc, CH2-hinge and CH3 domains; and engineeredFc-hinge domains with extended in vivo half lives, such as, for example,the mutant termed LSF. In addition, other immunoglobulin-like domainsmay be expressed employing the methods described therein.

[0396] Previous studies indicate that the CH2 domain may play animportant role in the control of catabolism of antibodies, and sequencesin the CH3 domain may be involved (Ellerson et al., 1976, Mueller etal., 1990; Pollock et al., 1990; Kim et al., 1994a: Medesan et al.,1997). The presence of carbohydrate residues on the CH2 domain appearsto have a minor if significant effect on the stability, and the extentof the effect is dependent on the isotype (Tao and Morrison, 1989).

[0397] Recombinant CH2-hinge, CH3, Fc and Fc-hinge fragments derivedfrom the murine IgG1 and human constant regions have been expressed fromhost cells. The CH3 domain, Fc fragment and Fc-hinge fragment were allfound to be homodimeric proteins. For the Fc and CH3 domain, the dimersare non-covalently linked, and are presumably stabilized by non-covalentinteractions. For the Fc-hinge dimer, the fragments are covalentlylinked by —S—S— bridges between the hinge region cysteines. Thesedomains may also be used to dimerize the adzyme target and catalyticdomains.

[0398] The immunoglobulin Fc-hinge and Fc fragments, purified followingexpression in host c ells, have the same in v ivo stability as a nativeantibody m olecule. Results from previous studies demonstrated that therecombinant aglycosylated Fc-hinge or Fc fragments have similarstability in vivo as the complete glycosylated IgG1 molecule. Therecombinant aglycosylated Fc-hinge fragment was found to have a 0 phasesimilar to that of a complete glycosylated IgG1 immunoglobulin molecule.In fact the removal of Fc-hinge resulted in a slight decrease in halflife (Kim et al., 1995). These results indicate that for the murine IgG1isotype the presence of carbohydrate residues does not appear to benecessary for in vivo stability, although it may still play a minorrole. Previous data obtained using protein chemistry suggested that theCH2 domain is responsible for in vivo stability (Ellerson et al. 1976)although a recent study indicated that residues in the CH3 domain mayalso be involved in the catabolism control of the murine IgG2a and IgG2bisotypes (Pollock et al., 1990).

[0399] (ii) Dosing Frequency

[0400] In many instances, an adzyme may be administered by injection oranother administration route that may cause some discomfort to apatient, or the adzyme may require the assistance of a physician orother medical professional for safe administration. In such instances,it may be desirable to design an adzyme that is therapeuticallyeffective at dosing frequencies of once per day or less, and preferablythe adzyme is effective when administered once per week, once every twoweeks, once every four weeks, once every eight weeks or less frequently.

[0401] The range of effective dosing frequencies for an adzyme maydepend on a variety of characteristics of the adzyme. For example, anadzyme with a shorter serum half-life will tend to be effective for ashorter period of time, leading to a more frequent dosing schedule.Various adzyme characteristics that can extend or decrease serumhalf-life are described above.

[0402] Drug reservoirs in the body may lengthen the time over which anadzyme is effective. Upon dosing, many drugs accumulate in bodycompartments, such as the fat reserves or various transcellular fluids,from which the drug is then released slowly over a long period of time.Similarly, a drug may be tightly bound by a serum protein, such asalbumin or alpha1-glycoprotein and thus retained in the serum in aninactive, protected bound form, from which it may be released slowlyover time. Accordingly, an adzyme may be designed to encourage theformation of reservoirs that provide for extended periods ofeffectiveness of the adzyme. In such embodiments, the adzyme may beadministered in a higher initial dose (a “loading dose”), followed byoccasional smaller doses (“maintenance doses”).

[0403] An adzyme may also be formulated and administered so as to havean extended period of effect. For example, an adzyme may be formulatedand administered to form a “depot” in the patient that slowly releasesthe adzyme over time. A depot formulations may be one in which theadzyme is encapsulated in, and released slowly from, microspheres madeof biodegradable polymers (e.g., polylactic acid, alginate). Other depotmaterials include gelfoam sponges and the ProLease® system (Alkermes,Inc., Cambridge, Mass.).

[0404] (iii) Selectivity

[0405] In many instances, an adzyme will be designed for delivery into aparticular milieu. For example, many adzymes for use in humans will bedesigned for delivery to and/or activity in the blood stream. Asdescribed herein, adzymes may b e designed for other situations, such asfor use in an industrial or environmental setting. In general, it willbe desirable to design an adzyme so as to decrease interactions withnon-target molecules that inhibit the effectiveness of the adzymeagainst the target, or, in other words, it will be desirable to designan adzyme that is active against target in the presence of expectedlevels of other components of the milieu in which the adzyme will beused.

[0406] In certain embodiments, an adzyme is designed to be effectiveagainst a substrate located in the blood, such as, for example, manyextracellular signaling molecules. Such an adzyme may be designed tominimize interactions with other blood components that would interferewith the ability of the adzyme to affect the target. The adzyme may beso designed on the basis of theoretical understanding or on the basis ofempirical study, or both. In certain embodiments, an adzyme retainseffectiveness against a target in the presence of one or more relativelyabundant blood components. An adzyme may be tested for activity againsttarget in the presence of one or more blood components, and particularlyabundant blood components. For example, an adzyme may be tested foractivity against target in the presence of one or more abundant serumproteins, such as serum albumin (e.g., human serum albumin or otherorganism-specific albumin), transthyretin (“retinol binding protein”),α-1 globulins (e.g., α-1 protease inhibitor [α-1 antitrypsin], α-1glycoprotein, high density lipoprotein [HDL]), α-2 globulins (α-2macroglobulin, antithrombin III, ceruloplasmin, haptoglobin),β-globulins (e.g., beta and pre-beta lipoproteins [LDL and VLDL], C3,C-reactive protein, free hemoglobin, plasminogen and transferrin),y-globulins (primarily immunoglobulins). In certain embodiments, anadzyme of the invention is active against target in the presence ofexpected (i.e., physiological, depending on the physiological state ofthe patient) concentrations of one or more blood components, such as oneor more abundant serum proteins. Optionally, the adzyme is activeagainst target in the presence of expected concentrations of an abundantserum protein, and optionally is not significantly affected byconcentrations of an abundant serum protein that are one-quarter,one-half, two, five or ten or more times greater than the expectedconcentration of an abundant serum protein. In a preferred embodiment,the adzyme comprises a catalytic domain that interacts with apolypeptide target that is expected to be found in the blood, andoptionally the catalytic domain has protease activity. Other abundantblood components include any of the various cell types, and moleculesfound on the surfaces thereof. Common blood cell types include red bloodcells, platelets, neutrophils, lymphocytes, basophils, eosinophils andmonocytes.

[0407] (vi) Resistance to Autocatalysis

[0408] In certain embodiments, the catalytic domain of an adzyme may beable to catalyze a reaction with the adzyme itself, resulting in thealteration of the adzyme. This type of reaction, termed “autocatalysis”may be between a catalytic domain and some other portion of the sameadzyme (e.g., a linker, targeting moiety or other part) or between acatalytic domain of one adzyme and a portion of a second adzyme (e.g.,the catalytic domain, linker, targeting moiety). The former will tend tobe more significant relative to the latter at very low adzymeconcentrations, such as may be expected to occur after an adzyme hasbeen deployed in a patient or other setting. The inter-adzyme form ofautocatalysis is most likely to occur at higher concentrations, such asduring adzyme preparation (e.g., purification from cell cultures andsubsequent concentration), storage and in any mixture prepared foradministration to a subject (e.g., a dose of adzyme mixed with salinefor administration intravenously).

[0409] For most types of catalytic domains, autocatalysis will be arelatively unimportant phenomenon, if it occurs at all. For example,catalytic domains that mediate glycosylation, isomerization orphosphorylation may not affect the activity of an adzyme, even if itdoes undergo autocatalysis. However, in certain situations, amodification of an adzyme could disrupt the ability of the adzyme to acteffectively on its target, particularly a modification that occurs inthe binding portion of an address moiety or in the active portion of acatalytic domain. Many types of catalytic domains require some type ofco-factor (e.g., ATP for a kinase, a sugar for a glycotransferase), andtherefore autocatalysis will not occur in the absence of suchco-factors. In these circumstances, autocatalysis may be avoided duringpreparation or storage by ensuring that there is little or no co-factorpresent in the adzyme preparation.

[0410] Catalytic domains that have protease activity or are otherwiseare capable of degrading the adzyme are of particular concern. Proteasesoften do not require any cofactor, and therefore autoproteolyticactivity may well occur at any stage of adzyme generation or use. Avariety of approaches may be taken to prevent autoproteolysis.

[0411] In one embodiment, an adzyme may be designed, or a proteasedomain selected, such that the protease is active at low levels in theabsence of the target. See, for example, the description of contingentadzymes provided herein.

[0412] In certain embodiments, protease vulnerable sites may beengineered out of the various portions of an adzyme, such as anypolypeptide address domain, catalytic domain or linker. This may beachieved either by altering the sequence of the selected components, orby selecting components in the first place that show resistance tocleavage with the desired protease domain. Trypsin has an internaltrypsin vulnerable site and is susceptible to trypsin action forinactivation, however trypsin-resistant trypsin mutants may begenerated. Often theoretic protease sensitive sites are present invarious domains but are not, in practice, viable protease substrates,perhaps due to folding or other steric hindrances. For example,Applicants have found that a p55(TNFR)-thrombin fusion protein adzymedoes not undergo autocatalytic proteolysis, despite the presence of athrombin cleavage site within the p55(TNFR) polypeptide. Such foldingmay be adjusted by the presence or absence of agents such as monovalentor divalent cations (e.g., potassium, calcium, zinc, iron) or anions(e.g., phosphates, chloride, iodine), as well as nonionic, zwitterionicand ionic detergents.

[0413] In certain embodiments, an address domain, such as a single chainantibody or other scaffold-based address domain, may be arrived at by invitro RNA selection. In vitro selection allows the selection forprotease insensitive address domains and thereby building an addressdomain that will not be cleaved by the enzyme domain. Similar approachesmay be used for linkers, immunoglobulin portions or other polypeptidesto be incorporated in an adzyme.

[0414] Anther means of limiting auto-proteolysis is to produce thecatalytic domain as a zymogen and activate the adzyme just prior to use(e.g., delivery to a patient). A zymogen or pro-protein portion may alsobe designed to be cleaved upon use (e.g., by a known serum activeprotease). Cleavage of certain zymogens occurs in the N-terminaldirection from the protease domain, meaning that after activation, theprotease domain will be separated from the portion of the polypeptidethat is N-terminal to the cleavage site. Cleavage of a zymogen thatoccurs in the N-terminal direction from the protease domain, means thatafter activation, the protease domain will be separated from the portionof the polypeptide that is N-terminal to the cleavage site. Cleavage ofcertain zymogens occurs in the C-terminal direction from the proteasedomain, meaning that after activation, the protease domain will beseparated from the portion of the polypeptide that is C-terminal to thecleavage site. Accordingly, a fusion protein comprising a zymogen shouldbe designed such that the protease domain is not separated (unless thatis the intent) from the other relevant portion of the fusion proteinupon activation.

[0415] In further embodiments, reversible competitive inhibitors may beemployed. Such inhibitors are preferably selected so as to be readilyremovable. An inhibitor for use in a pharmaceutical preparation may beselected to have a Ki that allows effective inhibition in the highconcentrations of storage and pre-administration, but which readilyreleases the protease upon dilution in the site o faction (e.g., in thepatient's body). Preferably, the inhibtor is chosen to be non-toxic orotherwise clinically approved. Inhibitors may also be used duringproduction and purification of adzymes. Many proteases require a metalcofactor, and such proteases can often be reversibly inhibited byformulation with a chelator, such as EDTA, EGTA, BHT, or a polyanion(e.g., polyphosphate).

[0416] In a further embodiments, protease vulnerable sites may bepost-translationally modified. Protease vulnerable sites could bemodified by phosphorylation or methylation or glycoyslation orchemically (in vitro, as opposed to modifications post translationallyduring production) such that the protease domain can not bind.

[0417] As a merely illustrative example, the competitive inhibitorbenzamidine has been used to block the action of trypsin in thetrypsinogen-p55 anti-TNF adzymes. The benzamidine has increased theyield of adzyme in the transient transfection expression of thetrypsinogen adzyme. Benzamadine, boronic acid or other proteaseinhibitors may be useful for manufacturing adzymes. With respect to theMMP7 catalytic domain, inhibitors such as Thiorphan, Ilomastat, FN 439,Galardin or Marimastat may be employed.

[0418] F. Exemplary Methods for Designing Adzymes

[0419] A significant advantage of adzymes is that they admits of anengineering and design approach that permits the biomolecular engineerto resolve several of the multiple engineering challenges inherent indrug design serially rather than simultaneously. A drug must not onlybind the target with high potency, but also it must have one or acombination of medicinal properties. In a given drug discovery/designexercise, the candidate molecule must exhibit various combinations ofthe following properties: a suitable solubility in blood, no significantinhibition of unintended targets (the higher specificity the better),achieve an effective concentration at the target, pass biologicalbarriers such as the skin, gut, cell walls, or blood brain barrier, haveno toxic metabolites, be excreted at a rate permitting achievement ofnecessary bioavailability without kidney or liver damage, not interferewith commonly prescribed medications, avoid complexation with albumin orother biomolecules or sequestration in tissue compartments, and besynthetically tractable. A single molecular entity simultaneouslydisplaying all necessary combinations of these properties is very hardto find or design.

[0420] In contrast, the individual molecular moieties that comprise theadzyme, e.g., the address and the catalytic domain, can be screenedindividually for the ability to bind to or modify the target ofinterest, respectively. Candidate structures for these parts can betaken from the ever growing public knowledge of new biological moleculesand and engineering efforts supported by increased understanding oftheir molecular biology and pharmacology. Existing active enzymes can bemutated to give them an address that will confer a new specificity.Nixon et al., in Proc. Natl. Acad. Sci. USA, Biochemistry Vol. 94,p.1069, 1997, have validated the approach of constructing an activeenzyme from disparate functional parts of other enzymes. Good candidatesfor each function may be linked together using various types of linkingstrategies. For example, they may be inserted into loops, attached viaflexible or structured amino acid sequence or other covalentattachments. Candidate constructs are made by choosing amino acidsequence or other structure spaced apart from the binding or catalyticportion of each domain for their ability to non-covalently complex, orvia candidate chaperone proteins that complex to both domains. It iscontemplated that many experimental constructs will be made in parallel,and that the library of constructs may be screened for desired activity,and active species evolved by mutagenesis or otherwise altered toexplore adjacent chemical space for improved properties.

[0421] Address domains can be selected using in vivo or in vitro assays.The address can be tested for the ability to bind to the target ofinterest using assays for direct binding or assays that measure theactivity of the target molecule. Methods that can be used to measurebinding of the address to the target molecule include biophysical andbiochemical techniques. For example, biophysical methods includefluorescence techniques which rely on intrinsic fluorescence or whichrely on the addition of an extrinsic label, e.g., fluorescence energytransfer, fluorescence anisotropy, changes in intrinsic fluorescence ofthe target molecule or address domain upon binding (see Lakowitz, J. R.(1983) Principles of Fluorescence Spectroscopy, Plenum Press, New York).Surface plasmon resonance (Sjolander, S. and Urbaniczky, C. (1991) Anal.Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol.5:699-705) can be used to study biospecific interactions in real time,without labeling any of the interactants (e.g., BIAcore). Changes in theoptical phenomenon of surface plasmon resonance can be used as anindication of real-time reactions between biological molecules.

[0422] Biochemical techniques that can be used to test the ability ofthe address domain to bind to the target molecule include techniquessuch as immunoprecipitation and affinity chromatography.

[0423] Further, one of both of the molecules can be labeled using aradioisotope, e.g., ¹²⁵I, ³⁵S, ¹⁴C, or ³H or other detectable label,e.g., an enzyme, and the interaction between the two molecules can bemeasured by specifically isolating one molecule and measuring the amountof the second molecule that is associated with the first molecule. Inthe case of a radiolabel, the amount of radio-labeled protein that isisolated can be measured by counting of radio emmission or byscintillation counting. Alternatively, compounds can be enzymaticallylabeled with, for example, horseradish peroxidase, alkaline phosphatase,or luciferase, and the enzymatic label detected by determination ofconversion of an appropriate substrate to product.

[0424] Address domains (e.g., a target specific peptide, target specificsingle chain antibody) may be taken from known examples in theliterature, preferably from examples of human proteins. Alternatively,address domains may be identified by any of a number of recombinantdisplay techniques, including but not limited to phage display, yeastdisplay, ribosome display, and bacterial display. Methods for preparingand screening libraries of address domains, e.g., peptide or antibodylibraries, are well known in the art and include those described in U.S.Pat. Nos. 6,156,511; 5,733,731; 5,580,717; 5,498,530; 5,922,545;5,830,721; 5,811,238; 5,605,793; 5,571,698; 5,223,409; 5,198,346;5,096,815; 5,403,484; 6,180,336; 5,994,519; 6,172,197; 6,140,471;5,969,108; 5,872,215; 5,871,907; 5,858,657; 5,837,242; 5,733,743;5,962,255; 5,565,332; and 5,514,548, the contents of each of which areincorporated herein by reference. Libraries may be functionally selectedor screened to identify specific address domains exhibiting the desiredproperties (e.g., affinity for a target, signal to noise ratio, etc.). Arecombinant display technique may also be used to identify candidateaddress domains. Useful recombinant display techniques include, but arenot limited to, phage display (see Hoogenboom et al., Immunol Today 2000August; 21(8):371-8), single chain antibody display (see Daugherty etal. (1999) Protein Eng 12(7):613-21; Makeyev et al., FEBS Lett 1999 Feb.12;444(2-3):177-80), retroviral display (see Kayman et al., J Virol 1999March;73(3): 1802-8), bacterial surface display (see Earhart, MethodsEnzymol 2000;326:506-16), yeast surface display (see Shusta et al., CurrOpin Biotechnol 1999 April;10(2):117-22), ribosome display (seeSchaffitzel et al., J Immunol Methods 1999 Dec. 10;231(1-2):119-35),two-hybrid systems (see, e.g., U.S. Pat. Nos. 5,580,736 and 5,955,280),three-hybrid systems, and derivatives thereof. Recombinant displaytechniques identify address domains capable of binding targets, e.g.,proteins (see, for example, Baca et al., Proc Natl Acad Sci USA 1997Sep. 16;94(19):10063-8; Katz, Biomol Eng 1999 Dec. 31;16(1-4):57-65; Hanet al., J Biol Chem 2000 May 19;275(20):14979-84; Whaley et al., Nature2000 Jun. 8;405(6787):665-8; Fuh et al., J Biol Chem 2000 Jul.14;275(28):21486-91; Joung et al., Proc Natl Acad Sci USA 2000 Jun.20;97(13):7382-7; Giannattasio et al., Antimicrob Agents Chemother 2000July;44(7): 1961-3).

[0425] Catalytic domains can be screened based on their activity.Depending on the specific activity of each molecule being tested, anassay appropriate for that molecule can be used. For example, if thecatalytic domain is a protease the assay used to screen the protease canbe an assay to detect cleavage products generated by the protease, e.g.,a chromatography or gel electrophoresis based assay. In an alternativeexample, the targeted substrate may be labeled and cleavage of thelabeled product may produce a detectable product by, for example, achange in fluorescence of the targeted substrate upon cleavage.

[0426] In another example, the catalytic domain may be a kinase. Theassay used to screen these catalytic domains could measure the amount ofphosphate that is covalently incorporated into the target of interest.For example, the phosphate incorporated into the target of interestcould be a radioisotope of phosphate that can be quantitated bymeasuring the emission of radiation using a scintillation counter.

[0427] It should be noted that the pharmacodynamics (binding and kineticproperties) of the interactions among the molecular address domains,targets, substrates, inhibitors, and enzymatically active sites willoften be important properties of candidate constructs embodying theinvention. Thus, association and dissociation properties, on-rates,off-rates, and catalytic reaction rates interplay in the variousconstructs to achieve the desired result. These properties areengineered into the molecules by a combination of rational, structurebased design and manufacture of a multiplicity of candidate constructs,or sub-parts thereof, which are screened for appropriate activity, asdisclosed herein.

[0428] Methods for preparing and screening catalytic domains for thedesired activity are well known in the art and described in, forexample, U.S. Pat. No. 6,383,775 and U.S. Provisional Patent ApplicationSerial No. 60/414,688, the entire contents of each of which areincorporated herein by reference.

[0429] Once the address domain and the catalytic domain have beenincorporated into a single molecule a library of adzymes may then becreated. The resulting library can be screened for the ability to modifythe specific target of interest. An assay for the appropriate biologicalfunction can be used to quantitate the amount of modification thecatalytic domain carries out. In a preferred embodiment, the catalyticdomain is a protease and the assay is one that measures the amount ofcleavage product generated by cleavage of the target molecule. It mayalso be effective to measure biophysical parameters, e.g., k_(cat) orK_(m), of the select library members. In another embodiment, the assayto screen the library of adzymes can be one which measures thebiological activity of the target molecule or a downstream molecule thatis regulated by the target molecule.

[0430] Once an adzyme, or group of adzymes, has been identified in aselection or screen, its properties may be further enhanced by one ormore rounds of mutagenesis and additional selection/screening accordingto art known methods. Furthermore, a catalytic domain of generalutility, such as a protease, may be used in constructs designed for verydifferent purposes.

[0431] A library of adzymes comprising combinations of address domains,linkers, and enzymes may be generated using standard molecular biologyprotocol. Either the address domain or the enzyme domain may be at theN-terminal of the adzyme. The size/length, composition (amino acidsequence) may be varied. Nucleic acids encoding the address domain, thelinker, and the enzyme domain can be recombinantly fused and cloned insuitable expression vectors, under the control of operatively linkedpromoters and transcription regulators. The construct may also includeepitope tags to facilitate purification of the recombinant products.

[0432] The desired combination of different address domain, linker, andenzyme domain can be generated, for example, by brute force constructionof a desired number of candidate adzymes. Each of these adzymes can thenbe individually tested and compared in one or more of in vivo and/or invitro functional assays, either for the adzyme itself, or for the targetof the adzyme, or both.

[0433] Once an adzyme, or group of adzymes, has been identified in aselection or screen, its properties may be further enhanced by one ormore rounds of mutagenesis and additional selection/screening accordingto art known methods. Furthermore, a catalytic domain of generalutility, such as a protease, may be used in constructs designed for verydifferent purposes.

[0434] To illustrate, U.S. Pat. No. 6,171,820 describes a rapid andfacilitated method of producing from a parental template polynucleotide,a set of mutagenized progeny polynucleotides whereby at each originalcodon position there is produced at least one substitute codon encodingeach of the 20 naturally encoded amino acids. Accordingly, the patentalso provides a method of producing from a parental templatepolypeptide, a set of mutagenized progeny polypeptides wherein each ofthe 20 naturally encoded amino acids is represented at each originalamino acid position. The method provided is termed “site-saturationmutagenesis,” or simply “saturation mutagenesis,” and can be used incombination with other mutagenization processes described above. Thismethod can be adapted to fine-tune/optimize the final chosen combinationof address domain, linker, and enzyme domain, so that the adzymeexhibits desired the biological property.

[0435] G. Contigent Adzymes

[0436] In one important class of adzymes, the activity of the catalyticdomain is modulated by the binding of the address to an address bindingsite (on the target or target associated molecule). Thus, the activityof the catalytic domain may be modulated by target itself, by a targetassociated molecule, or by part of the adzyme molecule itself. In thisclass of constructs, the catalytic domain itself is “masked” orsterically hindered, thus mostly inactive, when the address is not boundby an address binding site. Once the address recognizes and binds anaddress binding site (e.g., when the adzyme reaches its target), suchhinderance is released, exposing the active catalytic domain to act onthe target.

[0437] There could be many embodiments of this type of so-called“contingent adzymes.”

[0438] In one embodiment, the contingent adzyme is simply kept in aconformation that masks the catalytically active site, due to, forexample, the presence of a self inhibitory domain. Binding of theaddress to an address binding site triggers a conformation change of theadzyme, thereby releasing the masking effect of the catalytic domain.Alternatively, the masking of the catalytic domain may be released byadministering a molecule that binds to the self-inhibitory domain at atime and/or place where adzyme activity is needed. In fact, sometranscription factors have adopted this strategy to regulate theiractivity. For example, a small domain of the Drosophila homeodomaintranscription factor Bicoid (Bcd), located immediately N-terminal to thehomeodomain (residues 52-91), represses Bcd activity in Drosophilacells. This domain, referred to as a self-inhibitory domain, works as anindependent module that does not rely on any other sequences of Bcd andcan repress the activity of heterologous activators. This domain of Bcddoes not affect its properties of DNA binding or subcellulardistribution. A Bcd derivative with point mutations in theself-inhibitory domain severely affects pattern formation and targetgene expression in Drosophila embryos, suggesting that proper temporaland spacial regulation of the self-inhibitory domain is not onlypossible but crutial for certain biological functions. In fact, evidencesuggests that the action of the self-inhibitory domain requires aDrosophila co-factor(s), other than CtBP or dSAP18. These resultssuggest that proper action of Bcd as a transcriptional activator andmolecular morphogen during embryonic development is dependent on thedownregulation of its own activity through an interaction with a novelco-repressor(s) or complex(es) (Zhao et al., Development 129, 1669-1680,2002).

[0439] In another example, complement serine protease Factor D, which isessential for the activation of complement alternative pathway anddisplays a typical trypsin-like fold, contains an a typical active siteconformation due to the presence of a self-inhibitory loop, whichoverlaps with the binding positions of many peptidomimetic inhibitors inother serine protease complexes. Pro-factor D displays an activecatalytic triad conformation, flipped conformation for theself-inhibitory loop, and similar conformation for the flexibleactivation domain as that in trypsinogen, chymotrypsinogen, andprethrombin-2.

[0440] In another embodiment, the adzyme might be held in a conformationthat masks the catalytic domain by virtue of the presence of a selfbinding site (“SBS”) on the adzyme surface. The SBS may be within ornearby the catalytic domain, and it competes with the address bindingsite for binding to the address domain. In the absence of the addressbinding site, the otherwise enzymatically active catalytic site isinhibited sterically or allosterically by an intramolecular interactionbetween, for example, the address and the SBS, or between an attachedsmall molecule inhibitor and the SBS. In the presence of the addressbinding site, such as when the adzyme is at or near the target, thestability of the intramolecular interaction in the adzyme is reduced oreliminated, thereby increasing the activity of the enzymatically activecatalytic site to act on the target. Thus, in this class of embodiments,the catalytic domain of the adzyme is actuated, or turned on (exhibits ahigh reaction rate), or off (exhibits a lower reaction rate), dependingon the presence of a address binder for the address, which acts as aswitch or trigger. When the address binder is present, theintramolecular interaction is released (competed off), freeing up thecatalytic domain to act on its target.

[0441] One promonent feature of the contingent adzyme is that it has ahigher potential enzymatic activity in the locus of the target thanelsewhere, or when the adzyme activity is most needed. This constitutesa significant engineering advantage helpful in drug development, as itcan aid in the task of increasing specificity and decreasing toxicity,i.e., by reducing the chance that the construct reacts at unintendedtargets. In this type of construct, the affinity of the address for thetarget or target associated molecule preferably is greater than theaffinity of the address for the SBS.

[0442] In a related form of contingent adzyme, a small moleculeinhibitor that is mobile on a molecular scale is attached to thecatalytic domain by a flexible linker. Either the linker includes anaddress domain for the targeted biomolecule or other trigger molecule,or a portion of the catalytic domain apart from the catalytically activesite defines an address surface. In the absence of the trigger moleculeor targeted biomolecule, the inhibitor inhibits the enzymatic activityof the catalytic domain by inserting within and blocking access to itsactive pocket. In the presence of the trigger molecule or targetedbiomolecule, the address domain on the linker or the address surfacebinds with a binding site on the trigger molecule or targetedbiomolecule, and forms a complex which displaces the inhibitor away fromthe active pocket of the catalytic domain, increasing enzymaticactivity.

[0443] In still another form, an address domain is attached to thecatalytic domain via a linker which, in the absence of the binding siteon the target, sterically disposes the address domain in a position toimpede access to or otherwise disrupts the activity of the catalyticdomain. In the presence of the binding site, its association with theaddress domain disrupts the secondary structure of the linker,permitting the address domain to move away from the catalytic domain,freeing it up to react with its targeted biomolecule.

[0444] Several exemplary embodiments of the contingent adzymes aredescribed n more detail below. However, it should be understand thatthese illustrative examples are not exhaustive, and should not beconstrued to be limiting in any aspect. A skilled artisan would readilyappreciate the general principle of the invention and envision similarnon-described embodiments with minor modifications.

[0445]FIG. 3A is a schematic representation of an embodiment of acontingent adzyme comprising a single chain antibody (scFv) serving asan address domain, covalently attached by a flexible linker to acatalytic domain, which also defines an epitope or binding site for thescFv, termed a “self binding site” because the address of the constructis binding with the catalytic domain of the construct, i.e., engaging inan intramolecular reaction. In the absence of the targeted biomoleculeor other triggering molecule, the address binds to the self binding siteof the catalytic domain and thereby inhibits its catalytic activity.When the targeted biomolecule or other trigger molecule displaying thebinding site is present, it competes for binding to the scFv address,thereby freeing the catalytic domain to act on the targeted biomolecule,and increasing its activity, e.g., at least by a factor of 100, andpreferably by a factor of 103 to 109.

[0446] In environments of zero or low target concentration, the addressfavors binding to the self binding site on the catalytic domain, andtherefore the catalytic domain is inhibited from inducing reaction withspurious targets. In addition to the requirement that the address domainbind with both the self binding site on the catalytic domain and thetarget, and that the self binding site be positioned so as to interferewith activity of the catalytic domain when bound, it is preferred thatthe affinity of the address domain for the target be greater than itsaffinity for the binding site. In this case, thermodynamics favorsdisplacement of the address from the self binding site by the target,and promotes activation of the catalytic domain. Furthermore, it ispreferred that the off-rate of the target from the address domain behigh (fast) so that the mean dwell time of the address on its target iscomparable to the enzyme reaction time, preferably slightly longer, butnot a lot longer. Again, preferably the binding site on a targetundergoes a chemical or allosteric change when the target is convertedby the catalytic domain that decreases its affinity for the address.This promotes turnover.

[0447] The foregoing pharmacodynamic properties may be achievedconveniently by exploiting an antibody, antibody-like construct (scFv,etc.), or peptide which is selected, e.g., from a library, or designedto bind to a binding site on the targeted biomolecule. In this case, byepitope mapping or other conventional means, the sequence of amino acidsconstituting the binding site on the target can be determined, and thatsequence or a slightly altered, lower affinity sequence can beincorporated into various candidate positions in one or more loops of acatalytic domain. These positions are chosen based on the structuraldata characterizing the catalytic domain so as to maintain the structureof the enzymatic site, but to occlude or allosterically alter it whenthe inserted epitope is bound by the scFv or other address. Afterdetermination of which of the altered enzyme candidates preserve thecatalytic activity, the address, which binds both to the target and to aloop on the enzyme, is attached to the catalytic domain via a flexiblepolymeric leash or other flexible linear moiety. Again, variousconstructs are made for each of plural altered active enzymes, testingvarious linker lengths and attachment positions, until a structure isfound that exhibits inactivation or significant reduction in theactivity of the enzyme active site in the catalytic domain. This type ofconstruct, generally illustrated in FIG. 3A and following, has lowactivity in the absence of the target, as the scFv binds to the selfbinding site and sterically hinders the enzymatic site. In the presenceof the target, the competitive binding between the target and the scFvliberates the enzymatic site.

[0448]FIG. 3B is a schematic representation of another embodiment of acontingent adzyme comprising a small molecule or small peptide inhibitorattached covalently to a catalytic domain via a flexible linker thatincludes an address domain. The construct is enzymatically inactive(inhibited) in the absence or low concentration of a binder for theaddress domain as the inhibitor associates with the binding pocket ofthe catalytic domain, reducing or eliminating its activity. In thepresence of a binding site, here illustrated as being on a surface ofthe targeted biomolecule, the inhibitor is sterically displaced from theenzyme pocket freeing it to interact with the target. Such small peptidemay be similar in sequence to the enzyme binding domain of a substrate.The important feature is that the inhibitor is disposed within theenzyme site in the absence of the binder and spaced apart from it in itspresence, and that the address serves to increase the localconcentration of the target.

[0449]FIG. 3C is a schematic representation of another embodiment of acontingent adzyme similar to FIG. 3B comprising a small moleculeinhibitor (e.g., small peptide inhibitor) attached covalently to acatalytic domain via a flexible linker that includes an address domain.Again, the construct is enzymatically inactive (inhibited) in theabsence or low concentration of a binder for the address domain as theinhibitor associates with the binding pocket of the catalytic domain,reducing or eliminating its activity. In the presence of a binding site,here illustrated again as being on a surface of the targetedbiomolecule, the inhibitor is sterically displaced from the enzymepocket freeing it to interact with the target. In this case, anothermolecule of the targeted biomolecule, or one just released from theaddressd domain, reacts at the enzyme binding pocket. This type ofadzyme may be similarly constructed as those in FIG. 3B.

[0450]FIG. 3D is a cartoon of a contingent adzyme embodiment similar toFIG. 3A but showing an address domain non covalently complexed with acatalytic domain at a location comprising a self binding site whichinhibits substrate access to the enzyme pocket of the catalytic domain.The targeted biomolecule and its substrate site are here illustrated asone part of a two part protein complex. The address domain isillustrated as binding with a surface on the complex separate from thetargeted biomolecule, to form a three part complex. As illustrated, theportion of the address domain which binds with the catalytic domain toinhibit its enzymatic activity may be different from the portion whichbinds with the trigger molecule binding site. Furthermore, while theillustration depicts steric interference as the mechanism of catalyticinhibition, the address domain may allosterically inhibit the enzyme.This type of adzyme may be constructed by attaching to the addressdomain a catalytic domain binding sequence, such that when the addressand the catalytic domain is complexed via non-covalent interaction, theactive site on the catalytic domain is masked.

[0451]FIG. 3E is a schematic representation of still another embodimentof a contingent adzyme. In this embodiment, the interfering addressdomain is not bonded covalently or non covalently to the catalyticdomain, but rather to a chaperone protein that in turn is bonded to thecatalytic domain. The construct is enzymatically inactive (stericallyinhibited) in the absence of a binding site as the address domainassociates with the catalytic domain near the binding pocket, reducingor eliminating its activity. In the presence of a binding site, hereillustrated as being on a surface of the targeted biomolecule, theaddress domain is sterically displaced from the interfering locationnear the enzyme pocket, freeing it to interact with the target. Toconstruct this type of adzyme, a chaperon-binding domain may beincorporated into both the catalytic domain and the address tofacilitate chaperone binding. The small-molecule induced dimerizationsection of the instant specification describes various embodiments ofhow to bring together the address and the catalytic domain.

[0452]FIG. 3F is a schematic representation of still another embodimentof an contingent adzyme. Here, an address domain is non covalentlycomplexed with a catalytic domain through a chaperone protein. In theabsence of the target or other molecule recognized by the addressdomain, the activity of the catalytic domain is hindered by a smallmolecule inhibitor linked to the address domain and residing in theenzyme pocket. In the presence of the targeted biomolecule, the addressdomain is competed off the chaperone protein, extracting the inhibitorfrom the enzyme pocket. In the presence of the targeted biomolecule,there is competition between binding of the address domain to the targetand to the chaperone protein, displacing the small molecule, therebyfreeing the catalytic domain to act on the target.

[0453]FIG. 3G illustrates a contingent adzyme similar to the constructof FIG. 3B, but here a single globular protein domain is illustrated ascomprising both the address and the catalytic domain. In the presence ofthe triggering molecule displaying the binding site for the address, thesmall molecule inhibitor is competitively displaced from the bindingpocket of the catalytic domain, freeing it to induce chemical change inthe targeted biomolecule.

[0454] To test if a candidate contingent adzyme is relatively inactiveat the absence of the address binding site, while becoming activated atthe presence of the address binder, the apparent catalytic activity ofthe contingent adzyme can be measured at these two conditions, and theapparent catalytic activity at the presence of the address binder isexpected to be at least 2-fold, 3-fold, 5-fold, 10-fold, 20-fold,50-fold, 100-fold, or even more than that without the address binder.For example, the activity of a contingent adzyme may be measured at theabsence of any address binder, using a substrate that is incapable ofexposing the catalytic domain (such as a substrate that does not bind tothe address). To measure the activity of the contingent adzyme at thepresence of the address binder, the same substrate can be used, but thereaction system also contains an address binder which cannot itself beacted upon by the adzyme (such as resisting to proteolysis if the adzymeis a protease). In other words, the activity of the adzyme is comparedby the same substrate, with or without the presence of an address binderthat opens up the catalytic domain.

[0455] H. Methods of Treatment Using Adzymes

[0456] The present invention also provides a method of treating asubject suffering from a disease, such as a disease associated with asoluble or solvent accessible molecule. The method includesadministering to the subject a therapeutically, prophylactically, oranalgesically effective amount of an adzyme of the invention, therebytreating a subject suffering from a disease. Generally, adzymes can bedesigned and used for treating any disease mediated by a solventaccessible signaling factor in extracellular body fluid or on a cellsurface.

[0457] A disease associated with a soluble molecule includes a disease,disorder, or condition, which is caused by or associated (e.g., directlyor indirectly) with a soluble or membrane bound biomolecule, such as acytokine or a growth factor or a GPCR. Examples of such diseases includeinflammatory diseases, such as asthma, psoriasis, rheumatoid arthritis,osteoarthritis, psoriatic arthritis, inflammatory bowel disease (Crohn'sdisease, ulcerative colitis), sepsis, vasculitis, and bursitis;autoimmune diseases such as Lupus, Polymyalgia, Rheumatica, Scleroderma,Wegener's granulomatosis, temporal arteritis, cryoglobulinemia, andmultiple sclerosis; transplant rejection; osteoporosis; cancer,including solid tumors (e.g., lung, CNS, colon, kidney, and pancreas);Alzheimer's and other neurodegenerative disease; atherosclerosis; viral(e.g., HIV or influenza) infections; chronic viral (e.g., Epstein-Barr,cytomegalovirus, herpes simplex virus) infection; and ataxiatelangiectasia.

[0458] Adzymes may be used for anti-TNF therapies in place ofantibodies, artificial constructs, or small molecules. They can be usedto treat Wegner's vasculititus, Psoriasis, ankylosing spondylitis,Psoriatic arthritits, Crohn's and other IBD, and rheumatoid arthritis.They may also be used for routine or rapid intervention in infectiousdisease cased by bacteria and virus, attacking the infectious agentdirectly via cell surface proteins or circulating toxins or immunecomplexes. Examples of particularly promising soluble targets inaddition to TNF include IgE, C5, TGFβ, VEGF, and Interlukines such asIL-1.

[0459] Adzymes can be used in warm-blooded animals, preferably mammals,including humans. In a preferred embodiment, the subject is a primate.In an even more preferred embodiment, the primate is a human.

[0460] As used herein, the term “administering” to a subject includesdispensing, delivering or applying an adzyme of the invention e.g., anadzyme in a pharmaceutical formulation, to a subject by any suitableroute for delivery of the composition to the desired location in thesubject, including delivery by either the parenteral or oral route,intramuscular injection, subcutaneous/intradermal injection, intravenousinjection, buccal administration, transdermal delivery andadministration by the rectal, colonic, vaginal, intranasal orrespiratory tract route. The catalytic machines of the invention alsomay be administered by gene therapy approaches wherein nucleotidesencoding the constructs are administered to a patient, migrate or aretransported to target cells, enter the cells, and are expressed toprovide the cells with a therapeutic engineered intelligent machine.

[0461] The adzymes of the present invention can be provided alone, or incombination with other agents that modulate a particular pathologicalprocess. For example, an adzyme of the present invention can beadministered in combination with other known agents useful in thetreatment of diseases associated with or caused by a soluble molecule.Known agents that may be used in the methods of the invention can befound in Harrison's Principles of Internal Medicine, Thirteenth Edition,Eds. T. R. Harrison et al. McGraw-Hill N.Y., N.Y.; and the PhysiciansDesk Reference 50th Edition 1997, Oradell New Jersey, Medical EconomicsCo., the complete contents of which are expressly incorporated herein byreference. The adzymes of the invention and the additional agents may beadministered to the subject in the same pharmaceutical composition or indifferent pharmaceutical compositions (at the same time or at differenttimes). In one embodiment, one or more adzymes which are specific forone or more targets, are administered to a subject simultaneously. Inanother embodiment, the separate domains of the adzymes (i.e., theaddress domain and the catalytic domain) may be administered to asubject separately. In such an embodiment, the address domain and thecatalytic domain assemble in vivo to form the adzyme.

[0462] I. Non-medical Use of Adzymes

[0463] The present invention also provides various-uses of adzymes in anumber of non-medical applications, including but are not limited to,agriculture, environmental protection, food etc.

[0464] For example, the subject adzymes can find a wide range of uses inagriculture, including producing animal feed/pet food, grain milling,ethanol production, and food processing.

[0465] Animal Feed/Pet Food Adzymes may be used to upgrade nutritionalquality and removing anti-nutritional factors from feed components, suchas barley- and wheat-based feeds. Corn processing co-products such asgluten meal and fiber can also be improved using the subject adzyme. Infact, a number of food safty crisis in recent history (BSE, dioxinscare, etc.) have made it clear that animal feed has to be considered apublic hazard to public health and one that can lead to declining publiccondifence in the safety of food of animal origin.

[0466] In one embodiment, a protease may be linked to an addressspecific for the undesirable nutritional factor present in feedcomponents, thus leading to the degradation/elimination of suchcomponent. An added benefit of such adzyme-assisted digestion is thatthe degraded (inactive) protein factor is now a nutritious source ofprotein (peptide fragments). For example, Caughey et al. (J. Virol.2135-2141, Vol 68, No. 4, April 1994) reported that the apparentprecursor of protease-resistant PrP (responsible for the Prion diseaseBSE), protease-sensitive PrP, binds to Congo red and heparin, a highlysulfated glycosaminoglycan. Thus adzymes comprising an address domain ofcongo red or certain sulfated glycans, and an catalytic domain of aprotease that can degrade the protease-sensitive PrP, may be used topretreat certain animal feeds to reduce the risk of prion diseasetransmission.

[0467] In a related embodiment, adzymes of the instant invention may beused with other enzyme as feed additives to improve nutrition anddigestibility, reduce waste and lower feeding costs by increasing thesolubility of fibers or proteins from grains such as corn, wheat, barleyand soy used in typical feeds. The other enzymes that may be used withadzyme include: protease (protein-digesting enzyme), amylase(carbohydrate-digesting enzymes), lipase (fat-digesting enzyme),eellulase (fiber-digesting enzyme), lactase (milk sugar digestiveenzyme), invertase, maltase (sugar digestive enzyme), and/oralpha-galactosidase (bean digesting enzyme).

[0468] Fuel Ethanol Interest in ethanol as a clean-burning fuel isstronger than ever before. Fossil fuels are finite, nonrenewable andcause harm to the environment through pollution and global warming, yetthey supply over 80 percent of the world's energy needs. At the presentrate of consumption, world oil reserves are expected to deplete in thenext 50 to 100 years. Explosive population growth and improved livingstandards may shorten that timeframe. Ethanol, a chemical distilled fromstarch crops like corn, barley, sorghum and wheat, is both morefuel-efficient and far less polluting than gasoline. The agricultureindustry alone produces around 100 billion tons of biomass (unusedcrops, trees, grasses, and other agricultural “waste” product) worldwideeach year, with an energy value five times that of all energy consumedglobally. The starch from these grains is converted to fermentablesugars, which are then converted to alcohol by yeast.

[0469] While cellulase has been used in bio-fuel production, currentestimate for cellulase cost ranges from 30 to 50 cents per gallon ofethanol produced. To be more competitive in price, the objective inbio-fuel production is to reduce cellulase cost to less than 5 cents pergallon of ethanol. This requires a tenfold increase in specific activityor production efficiency or combination thereof. Thus even a few foldincrease in cellulase-specific activity (relative to the Trichodermareesei system) would be an important progress in this direction.

[0470] Thus in this embodiment, adzymes with cellulase and other enzymesthat can digest biomass into simpler suger moieties more suitable forfermentation may be used to facilitate the fuel ethanol production. Theaddress domain may contain more than one binding domains, such as theputative cellulose binding domains of 100 amino acids encoded by thecbpA gene of the Clostridium cellulovorans cellulose binding protein(CbpA). As mentioned before, the effective K_(d) of an adzyme with twoidentical address domains with K_(d) of about 1 nM would have a muchtighter value of about 10-15 M, and may increase the catalyticefficiency of cellulose digestion.

[0471] Food Processing Adzymes may be used in the food industry for suchpurposes as to improve baking, to process proteins more efficiently, topreserve foods, to treat animal hides in the leather industry, torecover silver residue in photographic film processing, and to improvepulp and paper processing. The use of adzymes offers treatmentalternatives that are less harsh than traditional chemical processes.The primary benefit offered by is treatments under mild conditions oftemperature and pH. Some adzymes useful for these purposes are used forprotein hydrolysis and in modification of cellulose and hemicellulose,others are useful to breakdown hydrogen peroxide in waste streams, orfor oxygen and glucose removal in food applications.

[0472] All these adzymes may be engineered, at the minimum, by includingmultiple address domains that may enhance the binding specificity and/oraffinity, thus increasing overall activity.

[0473] For example, adzymes can be used to improve gluten quality inbaked goods, enhance the sensory and physical characteristics of breads,and to facilitate the solubility, functionality and nutrition of meat orvegetable proteins in a diverse range of products from infant formula tosports drinks. Adzymes can also be used to more efficiently convertstarch to High Fructose Corn Syrup (HFCS), the sweetener widely used inmany foods and especially soft drinks.

[0474] Pulp and Paper Industry Adzyme comprising xylanases may be usedfor bleach boosting, adzyme comprising cellulases may be used forrefining pulp and paper recycling, and adzyme comprising amylases may beused for starch removal and modification.

[0475] In the brewing process, adzymes may be used to improve processefficiency and the final products. For example, adzymes comprising alphaamylases can be used in the cooking of cereal adjuncts, adzymescomprisin betaglucanases may be used to improve filtration in mashingand maturation, and adzymes comprisin glucoamylase can be used toproduce low calorie beers. In addition, adzymes comprisin alpha amylaseand glucomylase may be used in the production of potable alcohol.

[0476] Textile Industry Adzymes are useful in a variety of applicationswithin the cleaning and fabric care industries. The use of adzymes isbeneficial because they often replace chemicals or processes thatpresent environmental issues. But naturally occurring enzymes are quiteoften not available in sufficient quantities or enzymaticactivity/efficiency for industrial use.

[0477] The faded or worn look of denim that has a high contrast“stonewashed” look was originally achieved by washing denim with pumicestones in large industrial washing machines. In such a process, the lackof abrasion control, damage to the fabric, and wear and tear on thewashing machines is considerable. The same effects can be efficientlyachieved using adzymes, in a more environmentally friendly manner.Adzymes may be useful in desizing (amylases), denim finishing(cellulases), biofinishing of cotton and cellulosics (cellulases), andhydrogen peroxide elimination (catalases).

[0478] Personal Care Products Proteases perform various macromolecularmaturation or hydrolytic functions within the body. These functions maybe enhanced or modified by the application of exogenously suppliedadzymes. For example, applying adzymes to the skin's surface may aid inthe breakdown of surface oils and removal of dead skin. Thus, with theuse of adzymes in skin, hair and oral care applications, it is possibleto supplement the body's natural processes which will result in youngerlooking skin, more beautiful hair and healthier teeth and gums.

[0479] Detergent/Cleaner Products In other particularly preferredembodiments, the subject adzymes can be used to target and destroyparticular preselected molecules whether or not they have a biologicalactivity. Thus, for example, components of various soils or stains (e.g.milk, blood, eggs, grass stains, oil stains, etc.) can be specificallytargeted. For example, avidin/egg protein can be specifically targetedby using a biotin as a targeting moiety to specifically directed, e.g. aprotease to the site. The stain is degraded/digested and therebyreleased from the underlying substrate. Such adzymes are particularlyuseful in various cleaning formulations.

[0480] The term “soil” or “stain” refers to the accumulation of foreignmaterial on a substrate of interest (e.g. a textile). The “soil” or“stain” may have no biological activity, but may serve to discolor,and/or degrade the underlying substrate. The “soil” need not be visibleto the naked eye. Deposition of foreign materials that, while notvisible to the naked eye, but that create odors or support bacterialgrowth are also considered “soils” in the context of this application.Typical stains or soils include, but are not limited to grass stains,blood stains, milk stains, egg, egg white, and the like.

[0481] The adzymes of this invention are useful in a wide variety ofcontexts where it is desired to degrade a target molecule and/or inhibitthe activity of that target molecule. Thus, for example, in ex vivoapplications, the catalytic antagonists can be used to specificallytarget and degrade a particular molecule. Thus, for example, in cleaningoperations, the chimeric molecules of this invention can be utilized tospecifically target and degrade a component of a soil (e.g. a proteincomponent, a lipid component, etc.). In chemical synthetic processes, orbiochemical synthetic processes (e.g. in analytic or industrialpreparations, in bioreactors, etc.) to specifically degrade particularpreselected molecules. Thus, for example, where it is desired toeliminate a particular enzymatic activity in a bioreactor (e.g. aglycosylation) the catalytic antagonist of this invention comprises, asa targeting moiety, a substrate for the enzyme mediating the activity(e.g. a glycosyltransferase). The enzyme (receptor) in the reactor bindsthe targeting moiety and the enzymatic component of the chimera (e.g. ahydrolase) degrades the enzyme reducing or eliminating its activity andalso freeing itself from the enzyme binding site whereby it is free toattack another target enzyme.

[0482] Silicon Biotechnology

[0483] In biological systems, organic molecules exert a remarkable levelof control over the nucleation and mineral phase of inorganic materialssuch as calcium carbonate and silica, and over the assembly ofcrystallites and other nanoscale building blocks into complex structuresrequired for biological function (Belcher et al., Nature 381, 56-58,1996; Falini et al., Science 271: 67-69, 1996; Cha, Proc. Natl. Acad.Sci. USA 96: 361-365, 1999; Meldrum et al., Proc. R. Soc. Lond. B 251:238-242, 1993). This ability to direct the assembly of nanoscalecomponents into controlled and sophisticated structures has motivatedintense efforts to develop assembly methods that mimic or exploit therecognition capabilities and interactions found in biological systems(Colvin et al., J. Am. Chem. Soc. 144: 5221-5230, 1992; Brust et al.,Adv. Mater. 7: 795-797, 1995; Li et al., Chem. Mater. 11: 23-26, 1999;Alivisatos et al., Nature 382: 609-611, 1996; Mirkin et al., Nature 382:607-609, 1996; Brown, Proc. Natl. Acad. Sci. USA 89: 8651-8655, 1992).Brown (Proc. Natl. Acad. Sci. USA 89: 8651-8655, 1992; and NatureBiotechnol. 15: 269272, 1997) describes the successful selection ofpeptides with limited selectivity for binding to metal surfaces andmetal oxide surfaces. In another study, using combinatorialphage-display libraries, Whaley et al. (Nature 405: 665-668, 2000)extend this approach and successfully screened and selected numerouspeptides that bind to a range of semiconductor surfaces with highspecificity, depending on the crystallographic orientation andcomposition of the structurally similar materials used. Whaley et al.have extended this peptide recognition and specificity of inorganiccrystals to other substrates, including GaN, ZnS, CdS, Fe₃O₄, and CaCO₃.Such peptides may be used as the address domains of the subject adzymesand specifically place/assemble a variety of functional macromolecules(such as polypeptides) to pre-determined regions of micro-chips (such asprotein arrays, etc.).

[0484] Such targeted adzymes may be used for targeted, sequentialassembly/synthesis of nanomaterials, either inorganic ororganic/inorganic hybrid materials. In one embodiment, a first adzymemay be placed on the surface of a first region of a nano-assemblyline/container (tank, flow through tube, and other appropriate designs)such that the functional domain—the catalytic domain—on the adzyme maycarry out a first step of a series of processes on a reactant/substrate.The reactant can then be passed on to a second adzyme placed on thesurface of a second region of a nano-assembly line/container to allowthe next step of the process to finish. This process can be repeated forsubsequent steps of the processes until all the reactions are done andthe final product emerge.

[0485] The protein silicatein, which is isolated from the marine spongeTethya aurantia, catalyzes the in vitro polymerization of silica andsilsesquioxanes from tetraethoxysilane and silica triethoxides,respectively. When tethered to a specific region of the nanoassemblyline, the protein may be used to carry out specific steps of aparticular nanoassembly.

[0486] Alternatively, different adzymes may be attached to differentchips, which can be automatically loaded into or taken out of a reactioncontainer to carry out sequential catalytic steps.

[0487] Such targeted adzymes may also be used to construct arrays of(identical or different) molecules on silicon chips. In this regard, thesubject adzyme technology can be combined with planned chemical assemblyof 3-dimensional (3-D) organic/inorganic nanoscale architectures. Thisapproach is based on processes of surface chemical derivatization andcontrolled self-assembly taking place on organic template scaffoldsproduced via a hierarchical layer-by-layer self-assembly strategy. Forexample, arbitrary 2-D patterns can be generated using a novelnano-patterning process, referred to as “Constructive Nanolithography”[1], whereby electrical pulses delivered by a conductive AFM (atomicforce microscope) tip induce local electrochemical transformationsselectively affecting the top functions of certain highly orderedorganosilane monolayers or thicker films selfassembled on silicon. Inthis patterning process, the AFM tip plays the role of anano-electrochemical “pen,” with which chemical information is inscribedin a nondestructive manner on the top surface of the selected organicfilm. The patterned film or the product of its further chemicalmodification is then further utilized as a template capable of guidingthe subsequent surface self-assembly of various targeted adzymes, thuscreating a gradually evolving self-assembling system in which eachself-assembly step is subject to the control provided by a previouslyassembled template structure. This hierarchical self-assembly approachoffers options for the planned assembly of new types oforganic-inorganic nanocomposite architectures with variabledimensionality, from 0-D (individual dots) and 1-D (wires), to 3-D(superlattices) structures.

[0488] For example, the surface of an array may comprise a firstmaterial that cannot be bound by the addresses of any of a number ofadzymes later to be attached. Using the above-described technology, afirst area of the surface may be altered such that a first addressdomain of an adzyme may now bind to the altered region. If the bindingis saturating, after removing all of the first adzyme, the same processcan be repeated for a second region on the surface to expose a secondregion, such that a second adzyme (may be with a different catalyticdomain) may now bind. Since the ATM tip is controlling the exposure ofthe surface, various patterns can be etched on the surface sequencially,such that different adzymes with different functions may be selectivelyattached to different areas of the surface in distinct patterns ifnecessary.

[0489] DNA motifs have been used to produce nanoscale patterns in 2D,including a 2D lattice from a junction with sticky ends (see Seeman andBelcher, Proc Natl Acad Sci USA. 99 Suppl 2: 6451-5; Apr. 30, 2002; Epub2002 Mar 05). Thus in other embodiments, adzymes with address domainsrecognizing specific DNA sequences may be attached to such DNA latticeto create patterned adzyme arrays. The address domain can be naturallyexisting DNA binding domains, or can be selected for specific DNAbinding using, for example, phage display or other similar techniques.

[0490] J Compositions Containing Adzymes

[0491] (i) Protein Preparations

[0492] Another aspect of the invention pertains to pharmaceuticalcompositions containing the adzymes of the invention. The pharmaceuticalcompositions of the invention typically comprise an adzyme of theinvention or nucleotides encoding the same for transfection into atarget tissue, and a pharmaceutically acceptable carrier. As used herein“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and anti-fungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. The type of carrier can be selected basedupon the intended route of administration. In various embodiments, thecarrier is suitable for intravenous, intraperitoneal, subcutaneous,intramuscular, topical, transdermal or oral administration.

[0493] Pharmaceutically acceptable carriers include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersion. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the pharmaceuticalcompositions of the invention is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

[0494] Therapeutic compositions typically must be sterile and stableunder the conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, liposome, or other orderedstructure suitable to high drug concentration. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. In many cases, itwill be preferable to include isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, monostearate salts and gelatin. Moreover, theadzymes can be administered in a time release formulation, for examplein a composition which includes a slow release polymer. The adzymes canbe prepared with carriers that will protect the compound against rapidrelease, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, polylactic acid andpolylactic, polyglycolic copolymers (PLG). Many methods for thepreparation of such formulations are generally known to those skilled inthe art.

[0495] Sterile injectable solutions can be prepared by incorporating theadzyme in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the adzyme into a sterile vehicle which contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

[0496] Depending on the route of administration, the adzyme may becoated in a material to protect it from the action of enzymes, acids andother natural conditions which may inactivate the agent. For example,the adzyme can be administered to a subject in an appropriate carrier ordiluent co-administered with enzyme inhibitors or in an appropriatecarrier such as liposomes. Pharmaceutically acceptable diluents includesaline and aqueous buffer solutions. Enzyme inhibitors includepancreatic trypsin inhibitor, diisopropylfluoro-phosphate (DEP) andtrasylol. Liposomes include water-in-oil-in-water emulsions as well asconventional liposomes (Strejan, et al., (1984) J. Neuroimmunol 7:27).Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations may contain a preservative toprevent the growth of microorganisms.

[0497] The active agent in the composition (i.e., an adzyme of theinvention) preferably is formulated in the composition in atherapeutically effective amount. A “therapeutically effective amount”refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic result, such as modulationof the activity of a target, to thereby influence the therapeutic courseof a particular disease state. A therapeutically effective amount of anadzyme may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of the adzyme toelicit a desired response in the individual. Dosage regimens may beadjusted to provide the optimum therapeutic response. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the adzyme are outweighed by the therapeutically beneficial effects.In another embodiment, the adzyme is formulated in the composition in aprophylactically effective amount. A “prophylactically effective amount”refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired prophylactic result, for example,modulation of the activity of a target (e.g., TNFα or TNFβ) forprophylactic purposes. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

[0498] The amount of an adzyme in the composition may vary according tofactors such as the disease state, age, sex, and weight of theindividual. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

[0499] Another aspect of the invention provides aerosols for thedelivery of adzymes to the respiratory tract. The respiratory tractincludes the upper airways, including the oropharynx and larynx,followed by the lower airways, which include the trachea followed bybifurcations into the bronchi and bronchioli. The upper and lowerairways are called the conductive airways. The terminal bronchioli thendivide into respiratory bronchioli which then lead to the ultimaterespiratory zone, the alveoli, or deep lung.

[0500] Herein, administration by inhalation may be oral and/or nasal.Examples of pharmaceutical devices for aerosol delivery include metereddose inhalers (MDIs), dry powder inhalers (DPIs), and air-jetnebulizers. Exemplary nucleic acid delivery systems by inhalation whichcan be readily adapted for delivery of the subject adzymes are describedin, for example, U.S. Pat. Nos. 5,756,353; 5,858,784; and PCTapplications WO98/31346; WO98/10796; WO00/27359; WO01/54664;WO02/060412. Other aerosol formulations that may be used are describedin U.S. Pat. Nos. 6,294,153; 6,344,194; 6,071,497, and PCT applicationsWO02/066078; WO02/053190; WO01/60420; WO00/66206.

[0501] The human lungs can remove or rapidly degrade hydrolyticallycleavable deposited aerosols over periods ranging from minutes to hours.In the upper airways, ciliated epithelia contribute to the “mucociliaryexcalator” by which particles are swept from the airways toward themouth. Pavia, D., “LungMucociliary Clearance,” in Aerosols and the Lung:Clinical and Experimental Aspects, Clarke, S. W. and Pavia, D., Eds.,Butterworths, London, 1984. In the deep lungs, alveolar macrophages arecapable of phagocytosing particles soon after their deposition. Warheitet al. Microscopy Res. Tech., 26: 412-422 (1993); and Brain, J. D.,“Physiology and Pathophysiology of Pulmonary Macrophages,” in TheReticuloendothelial System, S. M. Reichard and J. Filkins, Eds., Plenum,New. York., pp. 315-327, 1985. The deep lung, or alveoli, are theprimary target of inhaled therapeutic aerosols for systemic delivery ofadzymes.

[0502] In preferred embodiments, particularly where systemic dosing withthe adzyme is desired, the aerosoled adzymes are formulated asmicroparticles. Microparticles having a diameter of between 0.5 and tenmicrons can penetrate the lungs, passing through most of the naturalbarriers. A diameter of less than ten microns is required to bypass thethroat; a diameter of 0.5 microns or greater is required to avoid beingexhaled.

[0503] An adzyme of the invention can be formulated into apharmaceutical composition wherein the compound is the only active agenttherein. Alternatively, the pharmaceutical composition can containadditional active agents. For example, two or more adzymes of theinvention may be used in combination.

[0504] (ii) Nucleic Acid Compositions

[0505] Another aspect of the invention provides expression vectors forexpressing the subject adzyme entities. For instance, expression vectorsare contemplated which include a nucleotide sequence encoding apolypeptide adzyme, which coding sequence is operably linked to at leastone transcriptional regulatory sequence. Regulatory sequences fordirecting expression of the instant polypeptide adzyme areart-recognized and are selected by a number of well understood criteria.Exemplary regulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology, Academic Press, San Diego, Calif.(1990). For instance, any of a wide variety of expression controlsequences that control the expression of a DNA sequence when operativelylinked to it may be used in these vectors to express DNA sequencesencoding the polypeptide adzymes of this invention. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, adenovirus or cytomegalovirus immediate earlypromoter, the lac system, the trp system, the TAC or TRC system, T7promoter whose expression is directed by T7 RNA polymerase, the promoterfor 3-phosphoglycerate kinase or other glycolytic enzymes, the promotersof acid phosphatase, e.g., Pho5, and the promoters of the yeast α-matingfactors and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof. It should be understood that the design of theexpression vector may depend on such factors as the choice of the targethost cell to be transformed. Moreover, the vector's copy number, theability to control that copy number and the expression of any otherprotein encoded by the vector, such as antibiotic markers, should alsobe considered.

[0506] As will be apparent, the subject gene constructs can be used tocause expression of the subject polypeptide adzymes in cells propagatedin culture, e.g. to produce proteins or polypeptides, includingpolypeptide adzymes, for purification.

[0507] This invention also pertains to a host cell transfected with arecombinant gene in order to express one of the subject polypeptides.The host cell may be any prokaryotic or eukaryotic cell. For example, apolypeptide adzyme of the present invention may be expressed inbacterial cells such as E. coli, insect cells (baculovirus), yeast, ormammalian cells. Other suitable host cells are known to those skilled inthe art.

[0508] Accordingly, the present invention further pertains to methods ofproducing the subject polypeptide adzymes. For example, a host celltransfected with an expression vector encoding a protein of interest canbe cultured under appropriate conditions to allow expression of theprotein to occur. The protein may be secreted, by inclusion of asecretion signal sequence, and isolated from a mixture of cells andmedium containing the protein. Alternatively, the protein may beretained cytoplasmically and the cells harvested, lysed and the proteinisolated. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The proteins can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins, includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for particular epitopes of the protein.

[0509] Thus, a coding sequence for a polypeptide adzyme of the presentinvention can be used to produce a recombinant form of the protein viamicrobial or eukaryotic cellular processes. Ligating the polynucleotidesequence into a gene construct, such as an expression vector, andtransforming or transfecting into hosts, either eukaryotic (yeast,avian, insect or mammalian) or prokaryotic (bacterial cells), arestandard procedures.

[0510] Expression vehicles for production of a recombinant proteininclude plasmids and other vectors. For instance, suitable vectors forthe expression of polypeptide adzymes include plasmids of the types:pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids,pBTac-derived plasmids and pUC-derived plasmids for expression inprokaryotic cells, such as E. coli.

[0511] A number of vectors exist for the expression of recombinantproteins in yeast. For instance, YEp24, YIp5, YEp51, YEp52, pYES2, andYRp17 are cloning and expression vehicles useful in the introduction ofgenetic constructs into S. cerevisiae (see, for example, Broach et al.,(1983) in Experimental Manipulation of Gene Expression, ed. M. InouyeAcademic Press, p. 83, incorporated by reference herein). These vectorscan replicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. Autotrophic selection or counterselection is often used inyeast. In addition, drug resistance markers such as ampicillin can beused in bacteria.

[0512] The preferred mammalian expression vectors contain bothprokaryotic sequences to facilitate the propagation of the vector inbacteria, and one or more eukaryotic transcription units that areexpressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo andpHyg derived vectors are examples of mammalian expression vectorssuitable for transfection of eukaryotic cells. Some of these vectors aremodified with sequences from bacterial plasmids, such as pBR322, tofacilitate replication and drug resistance selection in both prokaryoticand eukaryotic cells. Alternatively, derivatives of viruses such as thebovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. Examples of other viral (including retroviral)expression systems can be found below in the description of gene therapydelivery systems. The various methods employed in the preparation of theplasmids and transformation of host organisms are well known in the art.For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook,Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989)Chapters 16 and 17. In some instances, it may be desirable to expressthe recombinant polypeptide adzymes by the use of a baculovirusexpression system. Examples of such baculovirus expression systemsinclude pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors(such as the beta-gal containing pBlueBac III).

[0513] In yet other embodiments, the subject expression constructs arederived by insertion of the subject gene into viral vectors includingrecombinant retroviruses, adenovirus, adeno-associated virus, and herpessimplex virus-1, or recombinant bacterial or eukaryotic plasmids. Asdescribed in greater detail below, such embodiments of the subjectexpression constructs are specifically contemplated for use in variousin vivo and ex vivo gene therapy protocols.

[0514] Retrovirus vectors and adeno-associated virus vectors aregenerally understood to be the recombinant gene delivery system ofchoice for the transfer of exogenous genes in vivo, particularly intohumans. These vectors provide efficient delivery of genes into cells,and the transferred nucleic acids are stably integrated into thechromosomal DNA of the host. A major prerequisite for the use ofretroviruses is to ensure the safety of their use, particularly withregard to the possibility of the spread of wild-type virus in the cellpopulation. The development of specialized cell lines (termed “packagingcells”) which produce only replication-defective retroviruses hasincreased the utility of retroviruses for gene therapy, and defectiveretroviruses are well characterized for use in gene transfer for genetherapy purposes (for a review see Miller, A. D. (1990) Blood 76:271).Thus, recombinant retrovirus can be constructed in which part of theretroviral coding sequence (gag, pol, env) has been replaced by nucleicacid encoding a polypeptide adzyme of the present invention, renderingthe retrovirus replication defective. The replication defectiveretrovirus is then packaged into virions which can be used to infect atarget cell through the use of a helper virus by standard techniques.Protocols for producing recombinant retroviruses and for infecting cellsin vitro or in vivo with such viruses can be found in Current Protocolsin Molecular Biology, Ausubel, F. M. et al., (eds.) Greene PublishingAssociates, (1989), Sections 9.10-9.14 and other standard laboratorymanuals. Examples of suitable retroviruses include pLJ, pZIP, pWE andpEM which are well known to those skilled in the art. Retroviruses havebeen used to introduce a variety of genes into many different celltypes, including neural cells, epithelial cells, endothelial cells,lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/orin vivo (see for example Eglitis et al., (1985) Science 230:1395-1398;Danos and Mulligan, (1988) PNAS USA 85:64606464; Wilson et al., (1988)PNAS USA 85:3014-3018; Armentano et al., (1990) PNAS USA 87:6141-6145;Huber et al., (1991) PNAS USA 88:8039-8043; Ferry et al., (1991) PNASUSA 88:8377-8381; Chowdhury et al., (1991) Science 254:1802-1805; vanBeusechem et al., (1992) PNAS USA 89:7640-7644; Kay et al., (1992) HumanGene Therapy 3:641-647; Dai et al., (1992) PNAS USA 89:10892-10895; Hwuet al., (1993) J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S.Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

[0515] Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234,WO94/06920, and WO94/11524). For instance, strategies for themodification of the infection spectrum of retroviral vectors include:coupling antibodies specific for cell surface antigens to the viral envprotein (Roux et al., (1989) PNAS USA 86: 9079-9083; Julan et al.,(1992) J. Gen Virol 73:3251-3255; and Goud et al., (1983) Virology 163:251-254); or coupling cell surface ligands to the viral env proteins(Neda et al., (1991) J. Biol. Chem. 266: 14143-14146). Coupling can bein the form of the chemical cross-linking with a protein or othervariety (e.g. lactose to convert the env protein to anasialoglycoprotein), as well as by generating polypeptide adzymes (e.g.single-chain antibody/env polypeptide adzymes). This technique, whileuseful to limit or otherwise direct the infection to certain tissuetypes, and can also be used to convert an ecotropic vector in to anamphotropic vector.

[0516] Another viral gene delivery system useful in the presentinvention utilizes adenovirus-derived vectors. The genome of anadenovirus can be manipulated such that it encodes a gene product ofinterest, but is inactivate in terms of its ability to replicate in anormal lytic viral life cycle (see, for example, Berkner et al., (1988)BioTechniques 6: 616; Rosenfeld et al., (1991) Science 252: 431-434; andRosenfeld et al., (1992) Cell 68: 143-155). Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 d1324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled inthe art. Recombinant adenoviruses can be advantageous in certaincircumstances in that they are not capable of infecting nondividingcells and can be used to infect a wide variety of cell types, includingairway epithelium (Rosenfeld et al., (1992) cited supra), endothelialcells (Lemarchand et al., (1992) PNAS USA 89:6482-6486), hepatocytes(Herz and Gerard, (1993) PNAS USA 90:2812-2816) and muscle cells(Quantin et al., (1992) PNAS USA 89:2581-2584). Furthermore, the virusparticle is relatively stable and amenable to purification andconcentration, and as above, can be modified so as to affect thespectrum of infectivity. Additionally, introduced adenoviral DNA (andforeign DNA contained therein) is not integrated into the genome of ahost cell but remains episomal, thereby avoiding potential problems thatcan occur as a result of insertional mutagenesis in situations whereintroduced DNA becomes integrated into the host genome (e.g., retroviralDNA). Moreover, the carrying capacity of the adenoviral genome forforeign DNA is large (up to 8 kilobases) relative to other gene deliveryvectors (Berkner et al., supra; Haj-Ahmand and Graham (1986) J. Virol.57:267). Most replication-defective adenoviral vectors currently in useand therefore favored by the present invention are deleted for all orparts of the viral E1 and E3 genes but retain as much as 80% of theadenoviral genetic material (see, e.g., Jones et al., (1979) Cell16:683; Berkner et al., supra; and Graham et al., in Methods inMolecular Biology, E. J. Murray, Ed. (Humana, Clifton, N.J., 1991) vol.7. pp. 109-127). Expression of the inserted chimeric gene can be undercontrol of, for example, the E1A promoter, the major late promoter (MLP)and associated leader sequences, the viral E3 promoter, or exogenouslyadded promoter sequences.

[0517] Yet another viral vector system useful for delivery of thesubject chimeric genes is the adeno-associated virus (AAV).Adeno-associated virus is a naturally occurring defective virus thatrequires another virus, such as an adenovirus or a herpes virus, as ahelper virus for efficient replication and a productive life cycle. (Fora review, see Muzyczka et al., Curr. Topics in Micro. and Immunol.(1992) 158:97-129). It is also one of the few viruses that may integrateits DNA into non-dividing cells, and exhibits a high frequency of stableintegration (see for example Flotte et al., (1992) Am. J. Respir. Cell.Mol. Biol. 7:349-356; Samulski et al., (1989) J. Virol. 63:3822-3828;and McLaughlin et al., (1989) J. Virol. 62:1963-1973). Vectorscontaining as little as 300 base pairs of AAV can be packaged and canintegrate. Space for exogenous DNA is limited to about 4.5 kb. An AAVvector such as that described in Tratschin et al., (1985) Mol. Cell.Biol. 5:325135 3260 can be used to introduce DNA into cells. A varietyof nucleic acids have been introduced into different cell types usingAAV vectors (see for example Hermonat et al., (1984) PNAS USA81:6466-6470; Tratschin et al., (1985) Mol. Cell. Biol. 4:2072-2081;Wondisford et al., (1988) Mol. Endocrinol. 2:32-39; Tratschin et al.,(1984) J. Virol. 51:611-619; and Flotte et al., (1993) J. Biol. Chem.268:3781-3790).

[0518] Other viral vector systems that may have application in genetherapy have been derived from herpes virus, vaccinia virus, and severalRNA viruses. In particular, herpes virus vectors may provide a uniquestrategy for persistence of the recombinant gene in cells of the centralnervous system and ocular tissue (Pepose et al., (1994) InvestOphthalmol Vis Sci 35:2662-2666)

[0519] In addition to viral transfer methods, such as those illustratedabove, non-viral methods can also be employed to cause expression of aprotein in the tissue of an animal. Most nonviral methods of genetransfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral gene delivery systems of the present inventionrely on endocytic pathways for the uptake of the gene by the targetedcell. Exemplary gene delivery systems of this type include liposomalderived systems, poly-lysine conjugates, and artificial viral envelopes.

[0520] In a representative embodiment, a gene encoding anadzyme-containing polypeptide can be entrapped in liposomes bearingpositive charges on their surface (e.g., lipofectins) and (optionally)which are tagged with antibodies against cell surface antigens of thetarget tissue (Mizuno et al., (1992) No Shinkei Geka 20:547-551; PCTpublication WO91/06309; Japanese patent application 1047381; andEuropean patent publication EPA-43075). For example, lipofection ofneuroglioma cells can be carried out using liposomes tagged withmonoclonal antibodies against glioma-associated antigen (Mizuno et al.,(1992) Neurol. Med. Chir. 32:873-876).

[0521] In yet another illustrative embodiment, the gene delivery systemcomprises an antibody or cell surface ligand which is cross-linked witha gene targeting moiety such as poly-lysine (see, for example, PCTpublications WO93/04701, WO92/22635, WO92/20316, WO92/19749, andWO92/06180). For example, any of the subject gene constructs can be usedto transfect specific cells in vivo using a soluble polynucleotidecarrier comprising an antibody conjugated to a polycation, e.g.poly-lysine (see U.S. Pat. No. 5,166,320). It will also be appreciatedthat effective delivery of the subject nucleic acid constructsvia-mediated endocytosis can be improved using agents which enhanceescape of the gene from the endosomal structures. For instance, wholeadenovirus or fusogenic peptides of the influenza HA gene product can beused as part of the delivery system to induce efficient disruption ofDNA-containing endosomes (Mulligan et al., (1993) Science 260-926;Wagner et al., (1992) PNAS USA 89:7934; and Christiano et al., (1993)PNAS USA 90:2122).

[0522] In clinical settings, the gene delivery systems can be introducedinto a patient by any of a number of methods, each of which is familiarin the art.

[0523] For instance, a pharmaceutical preparation of the gene deliverysystem can be introduced systemically, e.g. by intravenous injection,and specific transduction of the construct in the target cells occurspredominantly from specificity of transfection provided by the genedelivery vehicle, cell-type or tissue-type expression due to thetranscriptional regulatory sequences controlling expression of the gene,or a combination thereof. In other embodiments, initial delivery of therecombinant gene is more limited with introduction into the animal beingquite localized. For example, the gene delivery vehicle can beintroduced by catheter (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (e.g. Chen et al., (1994) PNAS USA 91: 3054-3057).

[0524] The invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and the Sequence Listing arehereby incorporated by reference.

EXAMPLES

[0525] The following examples are for illustrative purposes only, andshould not be considered limiting in any respect.

Example 1 Chemical Cross-Linking of Address and Enzyme Domains

[0526] An adzyme can be created in at least two ways: (A) by chemicalcrosslinking and (B) by recombinant DNA technology.

[0527] The cross linking is performed using techniques well known in theart. Briefly, the N-termini (or surface accessible lysines) of oneprotein domain are reacted with SPDP, while the N-termini (or surfaceaccessible lysines) of the other protein domain are reacted with SMCC.Subsequently, the two domains are allowed to react, thus, formingdisulfide bridges that join the domains. When linked in the foregoingmanner, the estimated distance between the two domains is approximately14 Å.

[0528] Glutaraldehyde may also be used to cross link N-terminus of oneprotein with the C-terminus of the other protein.

[0529] These and similar methods well known in the art of chemicalcross-linking can be used to link address (such as the scFv Ab mentionedbelow) with a catalytic domain (such as thrombin or its zymogen).

Example 2 A Model Adzyme Experimental System

[0530] In order to create an adzyme (e.g., a bifunctional protein) thatpreserves the functions of both domains (address domain and catalyticdomain) and confers greater target specificity, applicants designed thefollowing model adzyme experimental system using prethrombin as anenzyme domain and a single-chain antibody specific for the hemagglutininpeptide of influenza virus (HA) [18] as the address domain. Such anadzyme has heightened proteolytic activity on substrates bound by theaddress domain compared to the proteolytic activity of the enzyme domainalone. Proteolytic adzymes are expressed and purified as inactivezymogens. Frequently the zymogen has an amino terminal sequence thatblocks the catalytic site. Cleavage at a specific activation siteremoves the blocking peptide and leads to protease activation. To ensurethat activation does not uncouple the two domains of the adzyme, theenzyme domain is preferably positioned N-terminal to the address domain.The following examples describe the construction, expression andpurification (see below, FIGS. 4 & 5) of components that include theaddress domain alone, the enzyme domain alone and the ADYZME thatcoupled the address and enzyme domains through a flexible polypeptidelinker. Following a partial one-step purification, these recombinantproteins were activated and tested for proteolytic activity againstsubstrates that either contained or lacked a binding site for theaddress domain. Schematic model adzyme and individual components areshown in FIG. 4.

[0531] In FIG. 4, all components were assembled in the pSecTag2A vectorsystem (Invitrogen, Carlsbad, Calif.), which included an N-terminalleader peptide designed to enable secretion from a heterologousexpression system and C-terminal tandem myc and His₆ tags to enableimmunodetection and purification. The address domain was a single chainantibody (scFvαHA) derived from monoclonal antibody mAb26/9, whichrecognized an influenza virus haemaglutinin (HA) epitope DVPDYA (SEQ IDNO:) [18]. The enzyme domain was prethrombin (residues 315 to 622 ofhuman prothrombin; accession no. AAC63054)—a zymogen of thrombin thatcould be activated using Factor Xa. Address and enzyme domains wereconnected with a 15 amino acid linker ([GGGGS]₃, SEQ ID NO:). Whentested against a target containing DVPDYA (SEQ ID NO:) and a suboptimalthrombin cleavage site (e.g., GGVR, SEQ ID NO:), the thrombin domain inthe adzyme demonstrates accelerated cleavage because of the higher localconcentration of peptide achieved through binding to DVPDYA (SEQ ID NO:)by the scFv domain (the address domain).

[0532] Both N-terminal and C-terminal fusions of adzymes are createdwith a variety of tags (myc, His₆, V5). Different linker compositionsand lengths are used. For example, the following constructs may becreated: thrombin-tag-COOH; scFvαHA-tag-COOH; N-thrombin-linker-scFvαHA-tag-COOH; N-scFvαHA-linker-thrombin-tag-COOH;N-scFvαHA-linker-thrombin-linker-scFvαHA-tag-COOH; or constructs withtwo thrombin units in tandem along with scFv anti-HA.

[0533] Prethrombin and the single chain antibody directed against the HAepitope are cloned individually into the HindIII and XhoI sites of thepSecTag2A vector from Invitrogen to generate proteins that will besecreted into the medium for subsequent biochemical characterization.Prethrombin is the inactive form that is activated by Factor Xa orecarin. Prethrombin(G₄S)₃-scHA and scHA(G₄S)₃-prethrombin are assembledby overlap/recombinant PCR (using the oligos described in Table X below)and cloned into the pSecTag2A vector as HindIII and XhoI fragments. Theywill contain myc and His₆ as tags at the C-terminus. The slash showswhere the cleavage occurs in the signal peptide. The amino acid sequencefor Prethrombin(G₄S)₃ scFvαHA is:METDTLLLWVLLLWVPGSTG/DAAQPARPAVRSLMTATSEYQTFFNPRTFGSGEADCGLR (SEQ ID NO:) PLFEKKSLEDKTERELLESYTDGRIVEGSDAEIGMSPWQVMLFRKSPQELLCGASLISDRWVLTAAHCLLYPPWDKNFTENDLLVRIGKHSRTRYERNTEKISMLEKIYIHPRYNWRENLDRDIALMKLKKPVAFSDYIHPVCLPDRETAASLLQAGYKGRVTGWGNLKETWTANVGKGQPSVLQVVNLPIVERPVCKDSTRIRITDNMFCAGYKPDEGKRGDACEGDSGGPFVMKSPFNNRWYQMGIVSWGEGCDRDGKYGPYTHVFRLKKWIQKVTDQFGEGGGGSGGGGSGGGGSMEVQLLESGGDLVKPGGSLKLSCAASGFTFSTYGMSWVRQTPDKRLEWVATISNGGGYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARRERYDENGFAYWGRGTLVTVSAGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLFNSGKQKNYLTWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQNDYSHPLTFGGGTKLEIKPADAAPTARGGPEQKLISEEDLNSAVDHHHHHH*.

[0534] The amino acid sequence for scHA(G₄S)₃prethrombin as made frompSecTag2 is:METDTLLLWVLLLWVPGSTG/DAAQPARPAVRSLMEVQLLESGGDLVKPGGSLKLSCAAS (SEQ ID NO:) GFTFSTYGMSWVRQTPDKRLEWVATISNGGGYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARRERYDENGFAYWGRGTLVTVSAGGGGSGGGGSGGGGSDIVMSQSPSSLAVSVGEKITMSCKSSQSLFNSGKQKNYLTWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQNDYSHPLTFGGGTKLEIKRADAAPTGGGGSGGGGSGGGGSMTATSEYQTFFNPRTFGSGEADCGLRPLFEKKSLEDKTERELLESYIDGRIVEGSDAEIGMSPWQVMLFRKSPQELLCGASLISDRWVLTAAHCLLYPPWDKNFTENDLLVRIGKHSRTRYERNIEKISMLEKIYIHPRYNWRENLDRDIALMKLKKPVAFSDYIHPVCLPDRETAASLLQAGYKGRVTGWGNLKETWTANVGKGQPSVLQVVNLPIVERPVCKDSTRTRITDNMFCAGYKPDEGKRGDACEGDSGGPFVMKSPFNNRWYQMGIVSWGEGCDRDGKYGFYTHVFRLKKWIQKVIDQFGEARGGPEQKLISEEDLNSAVDHHHHHH*.

[0535] TABLE X Oligo Name Alternative name Sequence (5′ to 3′) Length TmPurpose B1 scHAfwdHindIII CCCGGAAGCTTAatggaggtgcagctgttg 30 56 Fwdprimer for amplifying ScHA for cloning into pSecrag2A using HindIII. Aadded after HindIII site to maintain reading frame. B2 scHArevXhoIacgcccCTCGAGCagttggtgcagcatcagc 31 56 Reverse primer for amplify- ingscHA for cloning into pSecTag2A using XhoI. C added prior to XhoI siteto maintain reading frame. B3 prethrombinfwdH3CCCGGAAGCTTAATGaccgccaccagtgagtac 33 58 Fwd primer for amplifyingprethrombin into pSecTag2A using HindIII. A added to keep frame afterHindIII. B4 prethrombinrevXhoI ggcccCTCGAGCctctccaaactgatcaatg 31 56 Revprimer to clone pre- thrombin into XhoI site of pSecTag2A. C added tokeep frame. B5 G4ScHAfwd tttggagagggaggcggtgggtctggtgggggcggtagtg 72 56Forward primer to introduce gcggaggtgggagcatggaggtgcagctgttg (G4S)3 at5′ end of scHA. B6 prethrombinG4Srevcacctccatgctcccacctccgccactaccgcccccacca 73 54 Reverse primer tointroduce gacccaccgcctccctctccaaactgatcaatg (G4S)3 tag at the 3′ end ofprethrombin. B7 G4Sprethrombinfwdgcaccaactggaggcggtgggtctggtgggggcggtagt 75 58 Fwd primer to amplify pre-ggcggaggtgggagcATGaccgccaccagtgagtac thrombin with (G4S)3 at 5′ end tocreate overlap with ScHA. B8 scHAG4SrevggtggcggtCATgctcccacctccgccactaccgccccc 75 56 Rev primer to amplify scHAaccagacccaccgcctccagttggtgcagcatcagc with (G4S)3 at 3′ end to createoverlap with G4Spre- thrombin.

[0536] Substrates tested include: S1, a high affinity epitope (DVPDYA,SEQ ID NO:) recognized by scFvαHA linked to the proteolytic target site(HAE-PT: NH₂-YPYDVPDYA-(SGSGS)₄-GGVR-p-nitroanilide, SEQ ID NO:); andS2, the proteolytic target alone (PT: NH₂-GGVR-p-nitroanilide). Othersynthetic peptide substrates were also made with variable binding andcleaving substrate sequences. The Thrombin cleavage sites were chosenbased on the teachings of Backes et al. (2000) Nature Biotechnology18:187-193. Alternate choices include Ile-Thr-Pro-Arg as the bestcleavage site and IleThr-Leu-Arg as a poor target.

[0537] Cleavage of the peptide bond between the Arg residue in thesubstrates and the p-nitroanilide by thrombin activity releases freep-nitroaniline (pNA), which has a yellow color visible byspectrophotometric monitoring at 405 nm.

[0538] 2.1. Production of Model Adzyme Components: Construction,Expression, Purification and Activation.

[0539] Components were constructed in the pSecTag2A vector, expressedtransiently in mammalian cells and purified from conditioned media asdescribed below.

[0540] Briefly, mammalian expression vector pSecTag2A (Cat. No. V90020;Invitrogen, Carlsbad, Calif.) was used as the backbone for allconstructs. Upstream of the polylinker is a murine Ig K-chain V-J2-Csignal peptide, and downstream are myc and His₆ tags, a TAA stop codonand a bovine growth hormone polyadenylation signal. Other notablefeatures of the vector are a cytomegalovirus (CMV) promoter to driveexpression of the inserted coding sequence and the selectable markerszeocin and ampicillin. cDNAs corresponding to individual components weregenerated by PCR and cloned directionally into the polylinker tomaintain the reading frame using HindIII at the 5′ end and XhoI at the3′ end. The address component (scFvαHA) was amplified from a plasmidtemplate containing the coding sequence of scFvαHA (engeneOS, Waltham,Mass.); prethrombin was amplified from the full length human cDNA clone(ResGen; Cat. no. FL1001), and; the adzyme was created by overlap PCRdesigned to insert a 15 amino acid linker (GGGGS)₃ (SEQ ID NO:) betweenthe N-terminal prethombin domain and the C-terminal address domain. Allconstructs were sequence confirmed.

[0541] Transient transfections were carried out with 2×10⁶ 293T cellscultured in T175 flasks using Fugene (Roche, Indianapolis, Ind.).Conditioned media from 6 flasks containing the secreted components wereharvested when expression reached maximum levels (day 4, 5 or7—depending on the construct), clarified and dialyzed against 50 mMNaH₂PO₄, 300 mM NaCl, 5 mM imidazole (buffer A) overnight at 4° C. withone change of buffer. For purification, the dialyzed supernatants wereincubated for 16 h at 4° C. with Ni-NTA (Qiagen, CA) resin (0.4 ml resinor 0.8 ml of slurry per 200 ml of the dialyzed supernatant). Theresultant slurry was spun at 600 g for 10 mins at 4° C. and thesupernatant was removed and saved as a “flowthrough” sample. Then resincontaining bound protein was re-suspended in 10 ml buffer A, washed 3times (10 minutes each at 4° C.) and the beads were manually loaded on a3 ml syringe fitted with 3 mm Whatman filter paper. Three elutions(0.5-1 ml each) were performed with 50 mM NaH₂PO₄, 300 mM NaCl, 1 Mimidazole. Eluted material was dialyzed into phosphate-buffered salineovernight for storage or into Tris-buffered saline+1 mM CaCl₂ buffer foractivation with Factor Xa.

[0542] As shown in FIG. 5, the model adzyme prethrombin-(GGGGS)₃-scFvαHA(SEQ ID NO:) was expressed transiently in 293T cells and conditionedmedia harvested on day 7. The material was processed and purified asdescribed above. Samples representing equivalent portions of eachfraction were loaded onto 4-20% polyacrylamide gels and electrophoresedin Tris-glycine-SDS buffer (Novex). Panel A. Western blot—followingelectrophoresis the gel was electroblotted to nitrocellulose membraneswhich were stained with an anti-myc antibody (Invitrogen, Carlsbad,Calif.). Lane (1) Load; (2) Flow through; (3) Wash 1; (4) Wash 3; (5)Elution 1; (6) Elution 2; (7) Elution 3; (8) Resin boiled in sampleloading buffer; (9) Cruz mol. weight marker (Santa Cruz Biotechnology,Santa Cruz, Calif.). Panel B: Silver-stained gel. Lane (1) startingmaterial; (2) Flow through; (3) Wash 1; (4) Wash 3; (5) molecular weightstandard SeeBlue Plus 2; (6) Elution 1; (7) Elution 2; (8) Elution 3;(9) Resin boiled in sample loading buffer; (10) molecular weightstandard SeeBlue Plus 2.

[0543] An example of an electrophoretic analysis of the model adzymepreparation is shown in FIG. 5. The secreted full-length adzyme wasdetected with an anti-myc antibody at ˜70 kDa (panel A) as expected.Based on the silver-stained gel in this analysis (panel B), theestimated adzyme purity is about 10-20%. The individual address andenzyme components produced in parallel had yields and purity similar tothe model adzyme (data not shown).

[0544] Purified adzyme components containing enzyme domains wereactivated using Factor Xa, which cleaves prethrombin at Arg 320 therebyreleasing a 49 amino acid light chain from the N-terminus and generatingthe active thrombin heavy chain of 259 amino acids. In the example shownin FIG. 6, the activation process by Western blot indicated thatactivation using Factor Xa reduced the molecular weight of the modeladzyme by ˜6 kDa as expected.

[0545] Specifically, purified prethrombin and adzyme components weredialyzed at 4° C. overnight against 50 mM Tris pH 8, 0.1M NaCl, 1 mMCaCl₂, then protein concentrations were determined. Activation wasperformed using biotinylated Factor Xa (Roche). Applicant adapted theprotocol to account for the estimated purity (˜10%) of prethrombin to beactivated, thus 1 μg biotinylated Factor Xa was used per 4.44% g totalprotein for 3h at room temperature. Following activation, thebiotinylated Factor Xa was removed using streptavidin beads suppliedwith the kit, and the activated components were analyzed by Westernblot, and used for biochemical studies (see below).

[0546] As shown in FIG. 6, a preparation of our model adzyme (see FIG.5) was analyzed by Western blot using the anti-myc antibody before andafter activation using Factor Xa: partially purified model adzymedialyzed into TBS (lane 1); Factor Xa activation reaction (lane 2);activation reaction following removal of Factor Xa (lane 3);streptavidin beads used for removal (lane 4); and Cruz molecular weightstandards (lane 5, Santa Cruz Biotechnology, CA).

[0547] These examples demonstrate that Applicants have developedreliable production methods for preparing and activating recombinantadzyme components. A typical preparation from 2 to 6 T175 flasks yielded2-3 mg of material recombinant protein. These materials were sufficientfor all of the analytical studies on biochemical function describedbelow.

[0548] 2.2. Characterization of Adzyme Binding And Enzymatic Activity.

[0549] To ensure a meaningful comparison of the address domain, enzymedomain and adzyme properties (see Table 1), Applicants completed aseries of control experiments designed to: 1) measure binding to atarget epitope; 2) compare activities with well-characterized standardsand; 3) normalize the proteolytic activity against control substrates.

[0550] Binding to a target epitope. This experiment assessed the bindingcharacteristics of the adzyme address domain. Applicants assessedbinding activity of various components using biotinylated peptides in asandwich ELISA format. Purified components were dialyzed against PBS,captured on plates coated with anti-myc antibody (mAb 9E10; Sigma), thenanalyzed by ELISA for binding to biotinylated target peptide(NH2YPYDVPDYAGSGDYKAFD, SEQ ID NO:), which contained the high affinityepitope (underline). Bound peptides were quantified using astreptavidin-horseradish peroxidase detection system (Quantablue;Pierce, Rockford, Ill.). The address domain alone and both the activatedand zymogen forms of the adzyme bound comparable levels of the peptideper mole. However the enzyme domain alone failed to bind measurableamounts of the peptide, as expected.

[0551] Model adzvme thrombolytic activity. Characterization of theproteolytic activity of the model adzyme helps to determine if eitherthe address domain or the polypeptide linker affected its enzymaticproperties.

[0552] Applicants compared the activities of the model adzyme against acommercially available thrombin preparation (Sigma, St Louis, Mo.) onstandard fluoro or colorimetric derivatives of the thrombin tripeptidesubstrate—tosyl-gly-pro-arg-(p-nitroaniline, pNA or amino methylcoumarin AMC, Sigma). Activity was monitored over a 5 min. time coursein a cuvette-based fluorometric assay that measured released fluorophoreAMC (excitation at 383 nm, emission at 455 nm) in a Perkin Elmer LS55fluorescence spectrophotometer. Based on a standard curve for free AMC,data obtained in terms of arbitrary fluorescence units vs time wereconverted into molecules of substrate hydrolyzed per unit time. Reactionvelocities were determined over a range of substrate concentrations(0-50 μM) and K_(m) values for the tripeptide substrate and weredetermined using a Line-Weaver-Burke plot. From these studies, it wasconfirmed that commercially available human thrombin and the activatedmodel adzyme had comparable K_(m) values for this standard substrate,4.2 μM and 3.9 μM, respectively, which were in good agreement withliterature values.

[0553] Second, Applicants determined the specificity constants(K_(cat)/K_(m)) of thrombin for the substrates S1 and S2. Bothsubstrates contain a thrombin cleavage site, and substrate S1 alsoincludes the high affinity epitope recognized by the anti-HA singlechain antibody. A significant difference in thrombin selectivity foreither S1 (HAE-PT) or S2 (PT) would require the selection of analternative control substrate. Applicants measured the proteolyticactivity of a standard human thrombin preparation (Sigma) at twodifferent concentrations (0.0033 NIH Units/ml and 0.01 NIH Units/ml)against a concentration range between 3 μM to 25 μM of fluorometricderivatives of the substrates S1 and S2. Applicants followed the sameprotocol that was utilized to determine K_(m) values for thetosyl-GPR-AMC substrate (see above). Values for K_(m) and V_(max) werecalculated from Line-Weaver-Burke plots. Active and total enzymeconcentration(E_(total) was determined from active site titration with D-Phe-Pro-Arg-ChloroMethylKetone (D-FPR-CMK), an irreversible active site inhibitor. These experiments provided the data for a calculation of the absolute enzyme concentration (E)_(total)) in 0.0033 NIH Units/ml and 0.01 NIH Units/ml of Sigma thrombinproteolytic activity. From these data, Applicants calculatedK_(cat)=V_(max)/E_(total), then derived the specificity constantsK_(cat)/K_(m) for the substrates as 8.9 μM⁻¹sec⁻¹ and 10.3 μM⁻¹sec⁻¹ forS1 and S2, respectively. The close match of these values indicated thatthrombin was acting at either substrate with equivalent specificity andproteolytic activity. Thus, the high affinity epitope has no effect onthrombin activity.

[0554] Normalization of proteolytic activity. Applicants needed toquantify the enzymatic activity of the model thrombin-(GGGGS)₃scFvαHAadzyme with reference to the standard human thrombin. The commerciallyavailable tripeptide tosyl-GPR-pNA (Sigma), which lacked the highaffinity HA binding site was used as substrate. Cleavage of the peptidebond following the Arg residue releases released the chromophorep-nitroaniline (pNA) which is visible at 405 nm. Applicants determinedthe relative proteolytic activity, in units of thrombin activity per ml,of adzyme components before and after activation with Factor Xa. FactorXa has no activity on the commercial substrate. Data from one suchexperiment are shown below in FIG. 7. This allowed normalization basedon enzymatic activity of the adzyme preparation and comparison ofequivalent activities for adzyme and native commercial thrombin againstsubstrate S1 and S2.

[0555] Specifically, as shown in FIG. 7, proteolytic activity wasdetermined in a plate format using varying amounts of test componentsagainst a commercially available enzyme standard (3.3 nM human alphathrombin, Sigma) by monitoring the release of pNA absorbance at 405 nmin a Spectramax plate reader (Molecular Devices). Based on a standardcurve for free p-nitroaniline, data obtained in terms of absorbanceunits vs. time were converted into molecules of substrate hydrolyzed permolecule of enzyme per unit time.

[0556] Results of this experiment showed that this modelthrombin-(GGGGS)₃scFvαHA adzyme preparation: 1) had no detectableactivity prior to activation and; 2) could be normalized against astandard thrombin preparation-in this case 5 μl/ml of the activatedmodel adzyme was equivalent to 3.3 nM (0.1 NIH U/ml) of thrombin. Activesite titration of activated samples with D-FPR-CMK provided independentverification of the normalization. Hence, the proteolytic activity foradzyme preparations were normalized relative to the thrombin standard.

[0557] In summary, these control experiments have shown that: 1) theaddress domain-mediated binding to the high affinity epitope and linkageof an enzyme domain did not interfere with binding activity; 2) theactivated model thrombin-(GGGGS)₃scFvαHA adzyme had a K_(m) valuecomparable to thrombin for a standard thrombin substrate; 3) thrombinhad equivalent specificity for substrates S1 and S2; 4) activation usingFactor Xa was required to obtain detectable proteolytic activity; and 5)Applicants were able to normalize the proteolytic activities of adzymepreparations relative to a commercial thrombin standard. This series ofcontrol experiments have provided the basis for testing and comparingthe adzyme and isolated components on substrates that contained orlacked a high affinity epitope for the address domain.

[0558] 2.3. Test of Adzyme Function.

[0559] Applicants have designed an adzyme, thrombin-(GGGGS)₃scFvαHA,comprising a prethrombin enzyme domain linked by a 15 amino acidpolypeptide to a single chain antibody to the HA epitope as the addressdomain. Thrombin does not bind or cleave the HA epitope but binds itstargeted substrate site GGVR (SEQ ID NO:), whether in the context of S1or S2, with the same affinity. The activated thrombin component of thethrombin-scFvαHA adzyme also binds the GGVR (SEQ ID NO:) of S1 with thesame affinity; however the adzyme concept predicts that thrombin coupledto the anti-HA antibody will bind to substrates containing the HAepitope with the typical higher affinities of antibodies and may affectthe adzyme reaction rate. It is predicted that the adzyme could haveheightened enzymatic activity compared to thrombin.

[0560] In the reaction velocity experiments using the substrates S1 andS2 with either thrombin or thrombin-(GGGGS)₃scFvαHA adzyme; it ispredicted that: 1) the address domain alone (A) would be inactive (−) onboth substrates; 2) the enzyme alone (B) and the adzyme (D) would haveequivalent (+) proteolytic activity on substrate S2, the thrombincleavage site alone; 3) the adzyme would be more active (+++) againstsubstrate S1 (S1 has both the high affinity epitope and the thrombincleavage site) than against substrate S2 or the enzyme alone againsteither substrate (+); and 5) a stoichiometric mixture (C) of theunlinked address domain and enzyme domain would be equivalent to theenzyme domain alone on both substrates (+) (see Table 1) and less thanthe adzyme. TABLE 1 Model thrombin-(GGGGS)3scFvαHA adzyme and componentstested against linear peptide substrates Substrate Test componentS1:HAE-PT S2:PT A scFvαHA − − B Thrombin + + C A + B + + DThrombin-(GGGGS)₃scFvαHA +++ +

[0561] Adzyme activity is driven by the address domain. The proteolyticactivities of the model adzyme (D) to thrombin alone (B) were comparedon substrates that either contained (on S1) or lacked (on S2) a highaffinity epitope for the address domain. Results of this experiment areshown below in FIG. 8.

[0562] Specifically, in FIG. 8, proteolytic release of pNA fromsubstrates S1 and S2 was followed by monitoring absorbance at 405 nmover a two minute time course in a quartz cuvette. Reactions werecarried out in thrombin running buffer (50 mM Tris-HCl pH 8, 0.1M NaCl,0.1% polyethylene glycol 8000) containing matched active enzymeconcentrations (3.3 nM) as determined in normalization experiments (seeFIG. 6). Reactions were initiated with the addition of substrate to 25μM.

[0563] Equivalent activities of the activated thrombin-(GGGGS)₃scFvαHAadzyme and activated commercial thrombin, as determined with thetoysl-GPR-pNA substrate and hence normalized, were tested against S1 andS2. As shown in FIG. 8, the reaction rate for both the adzyme andthrombin are the same on the S2 substrate which contains just thethrombin cleavage site as expected, since both the adzyme preparationshad been normalized to thrombin. However, as predicted, the model adzymeshowed increased activity towards substrate S1 which contained a highaffinity epitope in addition to the thrombin cleavage site. There is a2× increase in reaction rate. The presence of this high affinity epitopeon the substrate did not alter the activity of the thrombin alone. Inthe absence of activation the adzyme did not show detectable proteolyticactivity. Thus the enhanced activity of thrombin-(GGGGS)₃scFvαHA adzymeis driven by the presence of an address domain that directed the enzymeactivity to the substrate through binding a high affinity epitope.

[0564] Enhanced adzyme activity requires linkage of the address andenzyme domains. To determine if the enhanced adzyme activity requireslinkage of the address and enzyme domain on the same polypeptide chain(D), or whether a stoichiometric mixture of the address domain andthrombin (C) perform equally well, Applicants compared these twoproteolytic activities on substrate S1, which contained a high affinityepitope for the address domain. Data from this comparison are shown inFIG. 9.

[0565] Specifically, in FIG. 9, purified address domain scFvαHA was usedat 3.3 nM (concentration estimated based on Bradford assay and estimatedpercent purity from a Coomassie Blue stained gel).

[0566] The results of the experiment clearly show that mixing theindividual address domain and enzyme thrombin together did not producethe accelerated rate of proteolysis observed with the model adzyme.Interestingly, applicants noted that the mixture was slightly lessactive than thrombin. Perhaps the unlinked address domain interferedslightly with access to the site of proteolysis by thrombin. Further,the address domain alone showed no detectable activity. Thus linkage ofthe address and enzyme domains produced a cooperative benefit inproteolytic rate over a stoichiometric mixture of the separated domains.

[0567] These studies have supported and validated the predicted adzymefunction. The model adzyme design has preserved the functions of theindividual components AND produced a cooperative advantage over thestoichiometric mixture. The technology can be equally applied to producea proteolytic adzyme specific for a clinically relevant target protein,such as TNF-α or IL-1.

Example 3 Adzymes that Selectively Inactivates the Bioactivity of TNF-α

[0568] This example describes the construction and optimization ofadzymes that selectively inactivate the bioactivity of TNFα.

[0569] To illustrate, ninty-six (96) adzyme structures for selectivecatalytic inactivation of TNFα are designed, and at least half areconstructed using standard molecular biology techniques. These adzymestructures include combinations of just two enzyme catalytic domains,three address domains and sixteen linkers (including zero linker).

[0570] Specifically, the enzymes are: cationic trypsin and MMP7; theaddresses are: Sp55, Sp55_(—2.6), and scFv; the linkers are: linkerswith 0, 10, 20, 30, 40, or 50 amino acids (corresponding to repeatingunits of GGGGS), FcIgG1 (knob mutation), FcIgG1 (hole mutation), FcIgG2(knob mutation), FcIgG2 (hole mutation), FcIgG3 (knob mutation), FcIgG3(hole mutation), FcIgG2-(G₄S)₂ hole mutation, FcIgG2-(G₄S)₄ holemutation, FcIgG2-(G₄S)₃ hole mutation, FcIgG2-(G₄S)₄ hole mutation. Theknob and hole mutations refer to the paired mutations(S354C:T366′W/Y349C:T366S:L368′A:Y407′V) in CH3 domains that had beenidentified as giving rise to predominantly heterodimeric bispecificantibodies (Merchant et al. Nature Biotechnology, 1998, 16, p. 677-681).

[0571] Six of the adzymes are then produced, purified, and tested forbioactivity. One or more of these adzymes fulfills the essentialcriteria of a useful adzyme—preserve the function of individualcomponents and yet produce a cooperative advantage through a polypeptidelinkage of the two domains. Specifically, the adzyme(s) inactivates TNFαmore effectively than either the address or enzyme alone, or astoiochiometric mixture of the individual domains.

[0572] Applicants have constructed, expressed and performed initialcharacterization of a series of three TNFα-targeted adzyme proteases,consisting of an address domain selected from soluble TNF receptor(s)linked to the catalytic domain of human cationic trypsin. The producedadzymes have been analyzed to quantify binding and proteolyticactivities. 3.1. Design of TNFα-specific Adzymes

[0573] Three components—the enzyme, the linker and the addressdomain—work together effectively to produce a catalytic antagonist ofTNFα. The enzyme domains are preferably positioned at the N-terminus inthis particular example, although in other adzyme designs, the enzymedomain may be C-terminal or even internal to the fusion protein. Theenzyme domain here is encoded as a zymogen and has proteolytic activitycapable of inactivating TNFα. The address domains will bind TNFα with ahigh degree of selectivity, and the linkers will produce functionalcoupling of enzyme and address domains to support cooperativity incatalytic inactivation of TNFα.

[0574] a. Selection of the enzyme domains A survey of the literature andpublic domain databases (MEROPS: http://www.merops.sanger.ac.uk) forproteases that are commercially available, expressible as zymogens, andexpected to cleave and inactivate TNFα [19-24] led to the selection oftwenty candidate proteases, which were then tested for inactivation ofTNFα using a TNF cytotoxicity assay. Specifically, TNF activation offunctional TNFα receptor TNFR-{[10, 25] leads to apoptotic cell death,which can be quantified in a cell-based assay [26]. This assay served asthe basis to screen the 20 proteases for inactivation of TNFαbioactivity (see below, FIG. 10, Table 2).

[0575] Specifically, in FIG. 10, L929 mouse connective tissuefibroblasts (ATCC catalog # CCL-1) were used to bioassay cell deathinduced by TNFα with the CellTiter 96® AQueous One Solution CellProliferation Assay system from Promega (Madison, Wis.). This systemprovides a colorimetric assay method for determining the number ofviable cells. Briefly, for each test protease, a solution of 5 [M TNFαwas digested overnight at 37° C., then bioactivity was determined foreight serial dilutions of the digestion solution. Data are mean valuesof triplicate determinations at each dilution of TNFα. Examples of TNFαinactivation by trypsin and MMP7 are shown in the figure. Results fromthe tests on all twenty proteases are summarized in Table 2.

[0576] More specifically, 10,000 L929 cells per well were seeded in 96well plates and cultured in DMEM+10% FBS overnight in a humidified CO₂incubator. Actinomycin D was added to all wells (final concentration 1μg/mL) and a standard TNFα survival curve was generated by adding humanTNFα (RDI, Flanders, N.J.) to achieve final concentrations in the wellsranging from 100 μg/ml−1 μg/ml. Protease digestion samples of TNFα weresimilarly diluted and added to parallel rows of wells. Triplicatedeterminations were done for each dilution of TNFα. Following anovernight incubation in a humidified CO₂ incubator 20 μl of pre-mixedMTS/PES was added to each well and incubation continued for 2-4 hours at37° C. Metabolically active viable cells reduced the assay reagent(MTS/PES includes a tetrazolium compound) into a formazan product thatwas soluble in tissue culture media. Absorbance was read at 490 nm in aplate reader after 4 hr to determine the number of viable cells.Complete details of the protocol were provided in Promega TechnicalBulletin No. 245. TABLE 2 Proteases tested for inactivation of TNFα.Proteases that inactivated TNFα Proteases that did not inactivate TNFMT1-MMP (0.86) Furin Urokinase Plasmin MMP12 (0.65) Cathepsin GEnterokinase Kallikrein5 Tryptase (0.62) HIV Protease TACE ADAMTS4MT2-MMP (0.5) ADAM10 MMP3 MT5-MMP ELASTASE (1.45) MMP7 (1.22)CHYMOTRYPSIN (2.74) TRYPSIN (2.3)

[0577] TNFα was digested with test proteases in overnight incubations at37° C., then analyzed for bioactivity as described in FIG. 10. Twelveproteases had no activity against TNFα; eight had varying levels ofactivity. Numbers in parentheses reflect log reduction in TNFα activitycalculated at the 50% survival level from inactivation curves similar tothe ones shown in FIG. 10.

[0578] The survival curve for standard TNFα shows a steep reduction insurvival from 100 pg/ml to 10 ng/ml (FIG. 10). In the presence of ˜600pg/ml TNFα reference standard only 10% of the cells survive. This is incontrast to 40% and 70% survival for the equivalent dilution of TNFαdigested with MMP7 or trypsin, respectively. The curve for dilutions oftrypsin-digested TNFα showed a consistent shift to the right, indicatingthat the bioactivity of TNFα was reduced more than two logs compared tothe TNFα reference standard. Similar studies were done with all of theenzymes listed in Table 2, including MMP7 (FIG. 10). Chymotrypsin wasthe most active protease against TNFα (2.74 log reduction in TNFαbioactivity). However it also showed significant auto-degradation (notshown), which may be improved by eliminating autocleavage sites in theenzyme (see above). All of these enzymes are candidates for the enzymecomponent of anti-TNF adzymes.

[0579] b. Selection of the address domains. Address domains willpreferably bind TNFα with high specificity, high affinity and willpreferably be resistant to proteolytic cleavage by the catalytic domain.Quantitative models of how binding domains cooperate [27] and ourexperience with the thrombin model adzyme (above) suggested a range ofbinding affinities suitable for TNFα-specific adzymes. Address domainswill be derived from two independent sources that bind TNFα withK_(affinity) values in the nM range—the TNFR-1 p55 extracellular domainand a single chain antibody to TNFα obtained from Genetastix (San Jose,Calif.) or generated in house from standard display technologies.

[0580] The sp55 address domains were constructed from the full-lengthhuman ectodomain of TNFR-1, and its binding to TNFα was characterized.Briefly, human TNFR-1 encoded by the CD120A gene (accession no.NM_(—001065); IMAGE clone 4131360, Invitrogen, Carlsbad, Calif.) wasused as the template to amplify residues 30-211 in the TNFR-1ecto-domain (protein accession no. P19438) [28] to construct afull-length sp55. Alternative address domains that might be evaluatedmay include subdomains of sTNFR-1, such as sp55Δ4 (residues 22-167) [29]or sp55 domain 2.6 (residues 41-150) [30]. These subdomains are smallerthan the full ecto-domain, and hence might have reduced sensitivity toproteolytic degradation. Since a significant function of the addressdomain is to bind the target with high affinity, sp55 binding to TNFαwas quantified using an indirect ELISA format to validate the presenceof a functional address domain (FIG. 11).

[0581] Briefly, in FIG. 11, address domains were expressed transientlyin 293T cells and captured on Ni-NTA coated wells. Binding to TNFα wasquantified using the S-Tag Tag™ system (Novagen, Madison, Wis.). TheS-Tag™ system is a protein tagging and detection system based on theinteraction of the 15 amino acid S-Tag peptide with ribonucleaseS-protein, which is conjugated with horseradish peroxidase (HRP).Applicants constructed, expressed and purified a human TNFα fusionprotein that included an N-terminal S-Tag™, then used this reagent(S-TNF) to quantify binding activity of the sp55 address domains(vertical stripes). Background (control) binding of TNFα that lacks theS-tag is shown in the hatched boxes.

[0582] More specifically in FIG. 11, conditioned media, harvested andclarified by centrifugation, was diluted 1:10 into buffer (0.5% BSAFraction V, 0.05% Tween-20 in 1 X PBS pH 7.4). Expressed proteins werecaptured on Ni-NTA coated wells (H is Sorb plates, Catalog # 35061,Qiagen) for 1 h at room temperature with shaking and washed four timesin 0.05% Tween-20 in 1×PBS to remove un-bound materials. Binding to TNFαwas determined by adding 100 μL of S-TNF (or control TNFα) at 1 μg/mL inassay buffer per well, followed by incubation for 1 hr at roomtemperature with shaking. Plates were washed 4 times in 0.05% Tween-20in 1×PBS, then S-protein HRP (1:2000 in assay buffer at 100 μL/well,Novagen, Madison, Wis.) was added and incubated for 1 hr further at roomtemperature with shaking. A final wash step in 0.05% Tween-20 in 1×PBSwas done 4 times to remove the S-protein-HRP, then 100 μL HRP substratetetramethylbenzidine (TMB; Sigma T 4444, St. Louis, Mo.) was added perwell. Color was allowed to develop for 5-45 minutes, then absorbanceread at 370 nm in a Spectromax plate reader (Molecular Devices).

[0583]FIG. 11 shows a three-fold elevation in S-TNF binding (verticalstripes) compared to non-specific binding in control samples (control:S-TNF; conditioned media from mock transfected cells). Binding appearedto saturate at 6-12% of conditioned media in the assay, and the dilutionseries showed that binding was proportional to the amount of expressedsp55 added. TNFα that lacked the S-tag was not detected withS-protein-HRP (hatched boxes). These results showed that the expressedsp55 address domain can bind TNFα.

[0584] As an alternative to using sp55 as an address domain, oneanti-TNFα scFV antibody will be selected from a set of eighteen thatwere obtained from Genetastix (San Jose, Calif.). These scFV antibodieswere identified by Genetastix through use of their proprietarytechnology (www.genetastix.com) as having TNFα binding activity.Briefly, a human scFv cDNA library was produced from polyA RNA of humanspleen, lymph nodes and peripheral blood lymphocytes throughamplification of V_(H) and V_(L) sequences that were assembled in framewith a GAL4 activation domain (AD). The 18 scFvs were identified asbinding human TNFα-lexA DNA binding domain when co-expressedintracellularly in yeast. The Genetastix scFvs expression vectors wereobtained in the form of bacterial periplasmic expression vector pET25B(Novagen, Madison, Wis.). Standard recombinant DNA methods were used tosubclone the scFv coding sequences into the pSecTag2A vector. Theconstructs were then sequenced to verify the structures. These scFvanti-TNFα antibodies is expressed and purified as described for theprevious adzyme components, then analyzed for binding to TNFα. Anindirect ELISA is used for TNFα based on the S-Tag™ system (see above,FIG. 11) to identify one of the 18 scFvs that shows high affinitybinding to TNFα for use as an address domain. The selection of aspecific scFv is based on a ranking of their relative binding strengthsof the various structures. Further quantitative determinations ofbinding affinities for TNFα may be included once a prototype adzyme hasbeen identified.

[0585] c. Selection of the linkers A significant function of a linker isto connect a catalytic domain and an address domain in a fusion proteinto yield cooperative function. The linker lengths can be experimentallyinvestigated. Applicants found that a triple-repeat (or “3-repeat”) ofthe flexible pentapeptide GGGGS enabled a functional linkage of theenzyme and address domains. This linker can range in length from 23.60 Åin α-helical conformation to 50.72 A as an extended chain. The initialadzymes have been built with 0 amino acids as linker (to minimizeintramolecular digestion, 3 amino acids (AAA) and 20 amino acids (4repeats of G₄S). Additional linker lengths under construction are 2repeats of G₄S (10 amino acids), 6 repeats of G₄S (30 amino acids), 8repeats of G₄S (40 amino acids) and 10 repeats of G₄S (50 amino acids).Extended form α-helical form (GGGGS)₂ (SEQ ID NO:) 32.02 Å 15.96 Å(GGGGS)₄ (SEQ ID NO:) 64.04 Å 31.92 Å (GGGGS)₆ (SEQ ID NO:) 96.06 Å47.88 Å (GGGGS)₈ (SEQ ID NO:) 128.08 Å 63.84 Å (GGGGS)₁₀ (SEQ ID NO:)160.1 Å 79.8 Å

[0586] d. Adzyme Structures There are currently no reports in theliterature for heterologous expression of trypsin in mammalian cells.Thus, it might be prudent to express the zymogen form that could beactivated by enterokinase. Trypsinogen was thus cloned to be in framewith the leader sequence and N-terminal to the linker and address domainand in frame with the tandem myc-His₆ tags at the C-terminus. N-murineIgκ leader sequence-trypsinogen-0aa-sp55-myc-His6 tgn-0-sp55 N-murineIgκ leader sequence-trypsinogen-AAA-sp55-myc-His6 tgn-3-sp55 N-murineIgκ leader sequence-(G₄S)₄-trypsinogen-20aa-sp55-myc-His6 tgn-20-sp55

[0587] e. Self- or auto-proteolysis of the adzyme by the catalyticdomain For those adzymes that employ a protease as a catalytic domain,it will generally be preferable to generate an adzyme that is resistantto self- or auto-proteolysis, which may affect the integrity andactivity of the address domain, the catalytic domain or the linker.

[0588] Accordingly, potential address domains may be tested for theirsusceptibility to protease attack. If the set of potential proteases andaddress domains is sufficiently large then there are likely to becombinations in which the protease attacks the target but not theaddress domain. Thus it may be advantageous to generate a relativelylarge library of potential adzymes, and screen among these candidateadzymes for the optimal combination of address domain, linker, andenzyme domain. Single chain antibodies, due to their beta sheetstructure, may be more resistant by nature to protease action. Onceselected, the linkage arrangement of the address and enzyme domain canbe used to minimize auto-proteolysis. Increasing the rigidity of thelinker, limiting the degrees of freedom of each adzyme domain orapplying a linker domain that orients the address and enzyme towardtarget but away from each other is possible. Additionally, addressdomains may be designed on the basis of evolved protein scaffolds, suchas that of the single chain antibody, and such scaffolds may bere-engineered at vulnerable conserved positions to remove proteasesensitive sites by mutagenesis. Alternatively or in combination,protease sites within an address or linker region may be selectedagainst by using, for example, display evolutionary techniques.

[0589] Additionally, certain enzymes can undergo autolysis within theenzyme domain. For example, trypsin undergoes autolysis at R122. Theautolysis site can be mutated to prevent autolysis (for example, R122His a mutation in the human trypsin I gene which leads to inactivation ofthe autolysis pathway and thus overexpression of active trypsin leadingto hereditary pancreatitis [31]). Protease domains can be expressed aszymogens to minimize the level of auto-proteolysis and maintain theadzyme in an inactive form. Adzymes will be activated immediately priorto application, or adzymes could be stored with an inhibitor that blocksthe catalytic site that can be diluted away to render the adzyme active.

[0590] 3.2. Production of Adzymes

[0591] Recombinant adzymes may be generated using the pSecTag2A vectorsystem or any other equivalently functional system for transientexpression in mammalian cells. The adzymes can be purified, for example,from conditioned media by binding the His₆ tags to a nickel resin.Additional technical details are described in example section 3.1.a.,above. All adzyme constructs generated in this section have beensequence confirmed.

[0592] a. Adzyme construction In this particular example, the enzymedomain is a zymogen of human trypsin, although similar constructs usinghuman MMP7 are also obtained. Human trypsin I (cationic trypsin) isencoded by PRSS1 gene (Accession #NM_(—002769)). The catalytic domainand part of the propeptide of trypsinogen I is amplified (residues16-247) from IMAGE clones 3950350 and 394971 (Invitrogen, Carlsbad,Calif.) and cloned into pSecTag2A. Human MMP7 (accession no. BC003635)residues 18-267, encoding the activation peptide (18-94) and catalyticdomain (95-267) is amplified from IMAGE clone 3545760 (Open Biosystems,Huntsville, Ala.) and cloned into pSecTag2A (data not shown).

[0593] Also in this particular example, the address domain used is sp55,although other address domains such as scFV anti-TNFα antibody may alsobe used (both selected from a set of 18 potential candidates). All ofthese constructs when completed are verified by DNA sequencing.

[0594] The amino acid sequence of trypsinogen (tgn) is:METDTLLLWVLLLWVPGSTG↓DIAPFDDDDKIVGGYNCEENSVPYQVSLNSGYHFCGGSL (SEQ ID NO) INEQWVVSAGHCYKSRIQVRLGEHNIEVLEGNEQFINAAKIIRHPQYDRKTLNNDIMLIKLSSRAVINARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDSCQGDSGGPVVCNGQLQGVVSWGDGCAQKNKPGVYTKVYNYVKWIKNTTAANSTRGGPEQKLTSEEDLNSAVDHHHHHH*

[0595] The amino acid sequence of trypsinogen-Oaa-sp55 (tgn-0-sp55) asexpressed from pSecTag2A is:METDTLLLWVLLLWVPGSTG↓DIAPFDDDDKIVGGYNCEENSVPYQVSLNSGYHFCGGSL (SEQ ID NO) INEQWVVSAGHCYKSRIQVRLGEHNTEVLEGNEQFINAAKIIRHPQYDRKTLNNDIMLIKLSSRAVINARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDSCQGDSGGPVVCNGQLQGVVSWGDGCAQKNKPGVYTKVYNYVKWIKNTIAANSLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTRGGPEQKLISEEDLNSAVDFHHHHHH*

[0596] The amino acid sequence of trypsinogen-3aa-sp55 (tgn-3-sp55) asexpressed from pSecTag2A is:METDTLLLWVLLLWVPGSTG↓DIAPFDDDDKIVGGYNCEENSVPYQVSLNSGYHFCGGSL (SEQ ID NO:) INEQWVVSAGHCYKSRIQVRLGEHNIEVLEGNEQFTNAAKIIRHPQYDRKTLNNDIMLIKLSSPAVINARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDSCQGDSGGPVVCNGQLQGVVSWGDGCAQKNKPGVYTKVYNYVKWTKNTIAANSAAALVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCTJNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTRGGPEQKLISEEDLNSAVDHHHHHH*

[0597] The amino acid sequence of trypsinogen-20aa-sp55 (tgn-20-sp55) asexpressed from pSecTag2A is:METDTLLLWVLLLWVPGSTG↓DIAPFDDDDKIVGGYNCEENSVPYQVSLNSGYHFCGGSL (SEQ ID NO:) INEQWVVSAGHCYKSRIQVRLGEHNIEVLEGNEQFINAAKTIRHPQYDRKTLNNDIMLTKLSSPAVTNARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDSCQGDSGGPVVCNGQLQGVVSWGDGCAQKNKPGVYTKVYNYVKWIKNTIAANSAAAGGGGSGGGGSGGGGSGGGGSRLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTRGGPEQKLISEEDLNSAVD HHHHHH*

[0598] In addition, sp55 was also cloned into pSecTag in similarfashion. The amino acid sequence of sp55 as expressed from pSecTag2A is:METDTLLLWVLLLWVPGSTG↓DAAQPARRAVRSLVPHLGDREKRDSVCPQGKYIHPQNNS (SEQ ID NO:) ICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTRGGPEQKLISEEDLNSAVDHHHHHH*

[0599] The adzymes are constructed from the individual enzyme andaddress domains connected via the three different linkers using anoverlap PCR method; as was done for the model thrombin adzyme (seeprevious examples). The constructs have been verified by DNA sequencing.

[0600] b. adzyme Expression Transient expression in 293T cells arecarried out in T175 flasks. Benzamidine, a small molecule competitiveinhibitor of trypsin activity with a K_(i) of 18 μM, is added to a finalconcentration of 1 mM to stabilize trypsinogen and trypsinogen adzymeexpression. Conditioned media is harvested at 24 hour intervals, orallowed to accumulate upto 72 hrs.

[0601] An example of representative expression as analyzed by Westernblotting with anti-myc antibody is shown below in FIG. 12. The increasedintensity of anti-myc signal in lane 2 demonstrates the stabilizingeffect of the small molecule trypsin inhibitor, benzamidine. Adzymescontaining 0, 3 and 20 amino acids as the linker are expressed atsimilar levels (lanes 3-5) and are also stabilized by the presence ofbenzamidine. The myc reactive band is of the expected size ofapproximately 51 kDa. Finally, sp55 is also produced in comparableamounts to trypsinogen and expression is not affected by the presence ofbenzamidine.

[0602] In brief, equal volumes of conditioned media after accumulationof secreted protein for 24 hours post transfection were electrophoresedon 4-20% TGS (Novex) gels, electroblotted to nitrocellulose membrane andstained with anti-myc antibody.

[0603] c. adzyme Purification In one embodiment, His₆-nickel methodologyis the preferred method of purification. This method is rapid, simpleand available in either column format for large batches or in a 96 wellformat for parallel assay testing. However, many other alternativemethods of purification can be used. For example, one option could bebenzamidine sepharose column chromatography (Pharmacia, NJ), whichincorporates a protease inhibitor into the resin. Standardcharacterization of purified proteins will include Western analysis withanti-myc antibodies and silver-stained gels to assess purity andrecovery of the adzyme preparations. The produced adzymes may be furtheranalyzed to quantify binding and proteolytic activities.

[0604] d. Recombinant protein determination. In one embodiment, adzymesare constructed with a carboxy terminal tandem myc-His₆ tags. An ELISAmethod is developed to detect the c-myc tag for quantitating recombinantproteins bound to Ni-NTA on surfaces. This helps to normalize the amountof adzyme used in any biochemical analyses and bioassays.

[0605] The following method can be used to quantify heterologouslyexpressed proteins containing tandem myc and His₆ tags using a sandwichELISA approach. In summary, diluted conditioned medium containingrecombinant proteins are incubated in wells of Ni-NTA coated H is Sorbmicrotiter plates (catalog no. 35061 Qiagen, Valencia, Calif.) and thenreacted with anti-myc-HRP (catalog no. R951-25, Invitrogen, Carlsbad,Calif.). Bound recombinant material is then detected by incubation witha chromogenic substrate. A standard curve was established in parallelwith purified recombinant sp55 (independently quantified using acommercially available ELISA (catalog no. QIA98, Oncogene ResearchProducts, Madison, Wis.) containing tandem myc His₆ tag allowingquantification of captured material. Conditioned media from mocktransfected cells served as a negative control.

[0606] In brief, conditioned media from transfections was diluteddirectly into assay buffer (0.5% BSA Fraction V, 0.05% Tween-20 in 1×PBSpH 7.4) to a final volume of 100 μL/well. Known amounts of the standard,sp55, was serially diluted in similar fashion in assay buffer. Bindingof the His₆ tag of the recombinant proteins to the Ni-NTA surface wasallowed to proceed at room temperature for half an hour with slowshaking. Anti-myc-HRP was then added to all wells at a final dilution of1:1500 such that the final volume in the wells was 150 μL. Binding wasallowed to proceed for two hours at room temperature with slow shaking.Following the binding of anti-myc to the His₆-captured proteins, thewells were washed 6 times with wash buffer (PBS containing 0.05% Tween20) and blotted dry. Then, the chromogenic substrate TMB (Sigma Catalog#T-4444) was added to each of the wells to a final volume of 100 μL. Theincrease in absorbance at 370 nm was monitored by a microtiter plateUV/VIS reader (Molecular Devices SPECTRAmax 384 Plus). All samples areassayed in duplicate.

[0607] Using this method of quantification, average yield of trypsinogenadzymes were estimated at 1 μg/mL.

[0608] 3.3. Biochemical Analysis of TNFα-Specific Adzymes.

[0609] This section describes methods to quantify binding andproteolytic activities of adzymes made against TNFα.

[0610] a. Adzyme binding Adzyme address domain functionality, e.g.,binding to TNFα, is quantified by the TNFα binding assay described aboveand by the ability of the address domain to independently inhibit TNFαactivity in the L929 assay prior to activation. Adzymes with theecto-domain of p55 have been tested with recombinant p55 as parallelcontrols. The adzyme proteins exhibit specific binding characteristics(amount of TNF bound per mole of protein) and binding affinities similarto the address domains alone.

[0611] Alternatively, the following method can be used to establish thepresence of a functional TNFα address domain within recombinantlyexpressed Adzymes by means of a modified ELISA-like assay. In summary,wells of a microtiter plate are precoated with TNFα and then reactedwith diluted conditioned medium containing candidate adzymes. Thedetection of adzymes that express a functional, high-affinity TNFαbinding domain (and hence, are retained on the microtiter platefollowing washing of the microtiter plate) is effected by subsequentcapture of a chromogenic enzyme conjugate that is specific for adetection tag within the adzyme and control constructs, followed byaddition of a chromogenic substrate. The inclusion of control wells inwhich the capture and detection of adzymes is not expected to bepresent, and parallel evaluation of similar constructs that do notencode the detection tag or TNFα-specific address domain providesevidence that the expressed adzymes contain a functional TNFα-specificaddress domain that binds specifically to the immobilized TNFα.Typically, one or more reversible or irreversible protease inhibitorsmay also be included in assay buffers to prevent autocatalysis orproteolytic activity of the adzyme, thereby restricting degradation ofthe adzyme and/or assay reagents.

[0612] In an illustrative example, an assay for humantrypsinogen-containing adzymes specific for TNFα is described. Wells ofa microtiter plate (Nunc-ImmunoModule, MaxiSorp Surface) were precoatedwith 100 μL/well of recombinant human TNFα: (RDI Catalog #RDI-301×) at aconcentration of 1 μg/mL diluted into phosphate-buffered saline (PBS),pH 7.2. An equal number of wells received 100 μL of PBS alone. Themicrotiter plate was then incubated at 4° C. overnight (approximately 16hours). The liquid from the wells was removed and the microtiter platewas washed twice with wash buffer (PBS containing 0.05% Tween 20). Allwells of the microtiter plate were blocked by addition of 200 μL/well ofblock/diluent buffer (PBS containing 0.05% Tween 20 and 0.05% bovineserum albumin [BSA; Fraction V, RIA & ELISA-grade, Calbiochem Catalog#125593]). The microtiter plate was incubated at room temperature for 2hours with slow shaking. The block solution was removed from the wellsand 100 μL/well of conditioned medium from transient adzymetransfections in 293T cells diluted 1:10 into block/diluent buffercontaining 1 mM benzamidine (Sigma Catalog #B-6506) was added toTNFα-containing wells and to wells that do not contain TNFα. The platewas incubated for 1 hour at room temperature with slow shaking.Following removal of liquid, the wells of the microtiter plate werewashed four times with wash buffer. Wells then received anti-mycantibody conjugated to horseradish peroxidase (anti-myc-HRP; InvitrogenCatalog #46-0709) diluted 1:2000 in block/diluent buffer containing 1 mMbenzamidine. The microtiter plate was incubated for 1 hour at roomtemperature with slow shaking. Following removal of liquid and washingas in the above, 100 μL/well of substrate (TMB, Sigma Catalog #T4444)was added to each of the wells. The increase in absorbance at 370 nm wasmonitored by a microtiter plate UVVIS reader (Molecular DevicesSPECTRAmax 384 Plus).

[0613] The results shown in FIG. 15 are from a representative experimentand reveal the mean OD and standard deviation for samples andexperimental controls evaluated in triplicate on a single microtiterplate. As illustrated, only Adzyme constructs and a control protein thatare able to bind to immobilized TNFα and that also contain the c-mycantibody sequence generate a positive signal above background at 370 nm.Included in this category is the trypsinogen-p55FL adzymes containing nolinker (Tgn-O-p55FL) as well as those containing linkers of 3 aminoacids (Tgn-3-p55FL) and 20 amino acids (Tgn-20-p55FL). As expected, apositive control sample containing the p55FL-myc-his construct (p55L)also binds and produces a positive signal above background. A constructconsisting of Trypsinogen-myc-his did not bind above backgroundpresumably due to significantly lower affinity for TNFα in the absenceof a high affinity address domain (p55FL). Similarly, the conditionedmedium from a transfection vector control (pSECTAG2A) did notdemonstrate a positive signal above background. Background, non-specificbinding of the anti-myc antibody to wells that contain or do not containTNFα was negligible as revealed by “Buffer Control.”

[0614] It should be understood that, although the present illustrativeexample detects binding to TNFα, this assay format is generic to any ofthe target molecules. One advantage of the assay described here is theinclusion of a reversible protease inhibitor in cell culture, during theexpression of the adzymes and in assay buffers, to prevent inadvertentautoactivation/proteolytic breakdown of the adzyme and/or activation byendogenous proteases. This can be used as a general solution toexpression of zymogens and/or active proteases. Importantly, one or moreprotease inhibitors can also be included in assay buffers for thepurposes of protein quantitation and confirmation of target specificity(as shown in this example). This general approach alleviates concernsregarding handling of autocatalytically-prone and/or active adzymes.

[0615] b. adzyme activation. Activation of the adzyme enzyme domain iscarried out by incubating at 37° C. following the manufacturer'srecommendations. The progress may be monitored by SDS-PAGE and Westernblotting (e.g., see FIG. 7). Enterokinase (Novagen, Madison, Wis.) wasused for activation of trypsinogen. For an in vitro TNFα assay,enterokinase need not be removed post activation, since it has beendetermined that enterokinase has no proteolytic activity towards TNFαand no effect in the L929 bioassay.

[0616] Applicants have developed a method for carrying out on-platecapture, activation and proteolytic assays for recombinantly producedenzymes or adzymes containing a His₆ tag. In summary, dilutedconditioned medium containing recombinant proteins are incubated inwells of Ni-NTA coated H is Sorb microtiter plates, then treated withenterokinase and presented with suitable peptide substrates. The peptidesubstrate used in the current example is tosyl-GPR-AMC (Catalog no.444228, Sigma, St. Louis, Mo.) which has been described previously.Proteolysis of the peptide bond between the Arg residue in the substrateand the AMC leads to the release of free fluorescent AMC (excitation 383nm, emission 455 nm). Inclusion of conditioned media from sp55 or vectortransfections provide important negative controls for the levels ofadventitious protease expression in transfected cells and substratebackground and hydrolysis under assay conditions.

[0617] In brief, conditioned medium containing recombinant proteins wasdiluted directly into assay buffer (0.5% BSA Fraction V, 0.05% Tween-20in 1×PBS pH 7.4) to a final volume of 100 μL/well. Typically, 5-25% ofconditioned medium per well yielded good linear response. Binding of theHis₆ tag of the recombinant proteins to the Ni-NTA surface was allowedto proceed at room temperature for two hours with slow shaking.Following the binding of anti-myc to the His₆-captured proteins, thewells were washed 6 times with wash buffer (PBS containing 0.05% Tween20 or PBST, 200 μL per wash) and blotted dry. This step alsoaccomplishes the removal of benzamidine which would otherwise interferewith subsequent steps in the assay. Activation of zymogen is achieved bythe addition of 1 U of enterokinase (EK, Catalog no. 69066, Novagen,Madison, Wis.) in a final volume of 100 uL of PBST. Activation wascarried out for 1 hour at 37° C. A parallel set of samples received noenterokinase but underwent similar incubation. Finally, the wells werewashed 6 times with PBST prior to the addition of trypsin digestionbuffer (100 mM Tris pH 8, 5 mM CaCl₂) containing 10 μM tosyl-GPR-AMC.Proteolytic activity was followed by monitoring the fluorescence at 455nm following excitation at 383 nm using a Gemini EM microplatespectrofluorometer (Molecular Devices, CA).

[0618]FIG. 13 shows a snapshot of representative experiments where thefluorescence detected at the end of 2 hours of incubation is comparedfor the different recombinant proteins. There is negligible proteolyticactivity in the absence of enterokinase activation of capturedrecombinant trypsinogen and trypsinogen adzymes (striped bars). In thisassay format, conditioned media from sp55 and vector transfections donot contain detectable amounts of proteases which could give rise toartifacts as evidenced by the background levels of fluorescence.However, following enterokinase treatment tryspinogen and the adzymes(tgn-O-p55, tgn-20-p55, tgn-3-p55) exhibit significant amounts ofproteolysis as evidenced by the 4-7 fold higher levels of fluorescenceas compared to the no activation controls.

[0619] On the other hand, MMP7 is activated with organomercurialcompound p-aminophenylmercuric acetate (APMA, Calbiochem 164610) andAPMA can be (and will be) removed according to instructions provided bythe supplier.

[0620] c. Proteolysis assay using synthetic peptide substrates. Theadzyme catalytic domain's proteolytic activity post activation wasdetermined with synthetic linear peptide substrates as described above.Proteolytic activity was determined in a plate format as described aboveusing varying amounts of adzymes and substrates against a commerciallyavailable enzyme standard. Substrate (tosyl-GPR-AMC) cleavage wasmonitored by the release of the fluorogen AMC. Data from arepresentative experiment is shown below in FIG. 14, where conditionedmedia from transfections (24 hours post transfection) were bound toNi-NTA plates, activated on plate, and assayed for proteolytic activitywith a fixed concentration (10 μM) of substrate (tosyl-GPR-AMC).

[0621] The assay for MMP7 proteolytic activity may use a fluorogenicsubstrate (dinitrophenyl-RPLALWRS; Calbiochem Cat. No. 444228).

[0622] Data from the biochemical analyses of adzymes can be used tonormalize the concentration and proteolytic activity of adzymepreparations for assessment of bioactivity.

[0623] 3.4. Testing Adzymes for Bioactivity.

[0624] To determine the bioactivity and selectivity of adzymes againstTNFa, adzymes will be used to inactivate TNFα and bioactivity will bequantified in a TNFα-induced L929 cell death bioassay. Selectivity canbe determined by comparing adzyme inactivation of TNFα alone and mixedwith human serum albumin (HSA). The soluble TNFα receptor p55 may serveas a stoichiometric blocker of TNFα.

[0625] The L929 bioassay is a stringent test for biologically activeTNFα. Assays are done using preparations of all twelve adzymes, plus thefour individual address and enzyme domains singly and in combinations.In each case, normalized quantities of purified adzymes (as assessedabove) will be mixed with TNFα alone or TNFα plus HSA and incubated at37° C. for 4 hr and overnight. The overnight digestion represents thestandard protocol. Preliminary results may be followed by time coursestudies as needed. Residual activity may be assayed by the L929bioassay.

[0626] It is expected that the enzyme domain a lone w ill inactivateTNFα and shift the survival curve to the right by 2 logs for the trypsindomain (FIG. 10, Table 2). In contrast, an effective adzyme will beexpected to effect a larger rightward shift and/or do so at much lowerconcentrations or more rapidly (e.g, 4 hr as opposed to overnight). A10-fold enhancement in the inactivation of TNFα (a shift in theinactivation curve one log unit to the right) is a convincingdemonstration of the potential of adzymes as catalytic proteinantagonists. Furthermore address domains alone should only minimallyinactivate (by stoichiometric binding) TNFα, and mixtures of the addressand enzyme domains should fare no better than the enzyme domains alone.The bioactivity of all adzymes may be ranked at matched molarconcentrations, and the selectivity of those that inactivate TNFα can beanalyzed.

[0627] Selectivity can be demonstrated in a mixing experiment (e.g., seeDavis et al, 2003)—adzymes will be used to digest TNFα alone and TNFαplus HSA, and the digests will be analyzed in the bioassay (see FIG.10). Human serum albumin is the most logical choice for this mixingexperiment. It is present in serum at high concentration and most likelyto pose a challenge to the selective action of a TNFα-specific adzyme.Initial tests of all adzymes can be done using a 10-fold molar excess ofHSA over TNFα. Adzymes that are not selective are expected to showreduced bioactivity in the presence of the competing substrate. Howeverselective adzymes should retain full bioactivity in the presence ofexcess HSA. Adzymes that pass this first test can be compared further byrepeating the analysis in the presence of a higher concentration of HSAin the mixture. Once again, adzymes can be ranked according to how muchbioactivity is retained in the presence of HSA. Several rounds ofcompetition should reveal structures that are both bioactive andselective catalytic antagonists of TNFα.

Example 4 Using Kinetic Modeling to Study the Adzyme System

[0628] Kinetic theory was applied to the reaction network of a directadzyme, shown in (Eq-2), to develop a mathematical model of adzymeperformance. Such a model can be used to design and optimize theparameters of an adzyme, and to predict important functional propertiesof the adzyme such as the amount of substrate that it can inactivate.

[0629] In this example, a simulation of the total amount of inactivationof a substrate by three different drugs was performed with the objectiveof comparing the potency of the adzyme to the potency of its constituentdomains individually. The three drugs were:

[0630] 1. An address with k_(on)=10⁶ M⁻¹s⁻¹ and k_(off)=10⁻³s⁻¹ (K_(D)=1nM)

[0631] 2. An enzyme with k_(on)=10³ M⁻¹s⁻¹, k_(off)=10⁻³ s⁻¹, andK_(cat)=1 s⁻¹ (K_(off)=10⁻³ M)

[0632] 3. A direct adzyme with the properties of the address and enzymeabove, and [S]_(eff)=10⁻⁶M.

[0633] The initial concentrations of the drugs were 50 pM and theinitial concentration of target substrate was 5 pM. The total amount ofsubstrate inactivated by each of these three drugs is shown in FIG. 16.

[0634] Specifically, FIG. 16 illustrates kinetic model results comparingthe performance of an adzyme, an address, and an enzyme. The resultsindicate that the adzyme inactivates significantly more substrate thaneither the address or the enzyme alone.

[0635] For example, the enzyme is too weak by itself to inactivate asubstrate at such low (pM) concentrations. Consequently, the totalamount of substrate inactivation by the enzyme is not significantlydifferent from zero. The address rapidly binds and inactivates somesubstrate, but because the concentration of substrate is much less thanthe K_(D) of the address, binding quickly becomes equilibrium limitedand the address can only inactivate about 0.25 pM, or 5%, of the totalsubstrate. The adzyme can rapidly bind and inactivate substrate like theaddress, but it can also convert the adzyme-substrate complex intoproduct, removing the equilibrium limitation.

[0636] This example shows that the model adzyme combines address andenzyme functionality in a synergistic way. Its potency is significantlyhigher than the sum of the address and the enzyme alone.

LITERATURE CITED

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[0638] 2. Zhou, P., Bogacki R., McReynolds L., Howley P M, Harnessingthe Ubiquitination Machinery to Target the Degradation of SpecificCellular Proteins. Molecular Cell, 2000. 6: p. 751-756.

[0639] 3. Bode, C., Runge M. S., Branscomb E. E., Newell J. B., MatsuedaG. R. and Haber E. Anitbody-directed Fibrinolysis. The Journal ofBiological Chemistry, 1989. 264(January 15): p. 944-948.

[0640] 4. Runge, M. S. B., Christoph; Matsueda, Gary R.; Haber, Edgar,Antibody-Enhanced Thrombolysis: Targeting of Tissue PlasminogenActivator in vivo. Proceedings of the National Academy of Sciences ofthe United States of America, 1987. 84(21): p. 76597662.

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[0642] 6. Zhou, H.-X., Quantitative Account of the Enhanced Affinity ofTwo Linked scFvs Specific for Different Epitopes on the Same Antigen. J.Mol. Biol. 2003: p. 1-8.

[0643] 7. Choy E H, P. G., Cytokine pathways and joint inflammation inrheumatoid arthritis. N Engl J Med., 2001. 344(12): p. 907-16.

[0644] 8. Feldmann M, M. R., Anti-TNF alpha therapy of rheumatoidarthritis: what have we learned? Annu Rev Immunol., 2001. 19: p. 163-96.

[0645] 9. Feldman, M., Development of anti-TNF therapy for rheumatoidarthritis. Nature Publishing Group, 2002. 2.

[0646] 10. Idriss, H. T. N., James H., TNFα and the TNF ReceptorSuperfamily: Structure-Function Relationship(s). Microscopy Research andTechnique, 2000: p. 184-195.

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[0649] 13. Nophar, Y., Brakebusch C., Englemann H., Zwang R., Aderka D.,Holtman H., Wallach D. Soluble forms of tumor necrosis factor receptors(TNF-Rs). The cDNA for the type I TNF-R, cloned using amino acidsequence data of its soluble form, encodes both the cell surface and asoluble form of the receptor. EMBO J, 1990. 9(10): p. 3269-78.

[0650] 14. Maini R N, Z. N., Rheumatoid arthritis, in Rheumatology, D.P. Klippel J H, Editor. 1994, Mosby: London. p. 3.1-3.14.8.

[0651] 15. Warris, A., A. Bjorneklett, and P. Gaustad, Invasivepulmonary aspergillosis associated with infliximab therapy. N Engl JMed, 2001. 344(14): p. 1099-100.

[0652] 16. Keane, J., Gershon S., Wise R. P., mirabilke-Levens E.,Kasznica J., Schwieterman W. D., Siegel J. N., Braun M. M. Tuberculosisassociated with infliximab, a tumor necrosis factor alpha-neutralizingagent. N Engl J Med, 2001. 345(15): p. 1098-104.

[0653] 17. Williams, R. O., M. Feldmann, and R. N. Maini, Anti-tumornecrosis factor ameliorates joint disease in murine collagen-inducedarthritis. Proc Natl Acad Sci USA, 1992. 89(20): p. 9784-8.

[0654] 18. Churchill M E, S. E., Pinilla C, Appel J R, Houghten R A,Kono D H, Balderas R S, Fieser G G, Schulze-Gahmen U, Wilson I A.,Crystal structure of a peptide complex of anti-influenza peptideantibody Fab 26/9. Comparison of two different antibodies bound to thesame peptide antigen. J Mol Biol., 1994. 241(4): p. 534-56.

[0655] 19. Calkins C C, P. K., Potempa J, Travis J., Inactivation oftumor necrosis factor-alpha by proteinases (gingipains) from theperiodontal pathogen, Porphyromonas gingivalis. Implications of immuneevasion. J Biol Chem, 1998. 273(12): p. 6611-4.

[0656] 20. Nakamura K, K. M., Proteolysis of human tumor necrosis factor(TNF) by endo-and exopeptidases: process of proteolysis and formation ofactive fragments. Biol Pharm Bull:, 1996. 19(5): p. 672-7.

[0657] 21. Narhi L O, R. M., Hunt P, Arakawa T., The limited proteolysisof tumor necrosis factor-alpha. J Protein Chem, 1989. 8(5): p. 669-77.

[0658] 22. Kim Y J, C. S., Kim J S, Shin N K, Jeong W, Shin H C, Oh B H,Hahn J H., Determination of the limited trypsinolysis pathways of tumornecrosis factor-alpha and its mutant by electrospray ionization massspectrometry. Anal Biochem., 1999. 267(2): p. 279-86.

[0659] 23. Magni F, C. F., Marazzini L, Colombo R, Sacchi A, Corti A,Kienle M G., Biotinylation sites of tumor necrosis factor-alphadetermined by liquid chromatography-mass spectrometry. Anal Biochem.,2001. 298(2): p. 181-8.

[0660] 24. van Kessel K P, v. S. J., Verhoef J., Inactivation ofrecombinant human tumor necrosis factor-alpha by proteolytic enzymesreleased from stimulated human neutrophils. J. Immunol., 1991. 147(11):p. 3862-8.

[0661] 25. Locksley R M, K. N., Lenardo M J., The TNF and TNF receptorsuperfamilies: integrating mammalian biology. Cell, 2001. 104(4): p.487-501.

[0662] 26. Humphreys, D. T. and M. R. Wilson, Modes of L929 cell deathinduced by TNF-alpha and other cytotoxic agents. Cytokine, 1999. 11(10):p. 773-82.

[0663] 27. Zhao, X. M., L; Song, K; Oliver, P; Chin, S Y; Simon, H;Schurr, J R; Zhang, Z; Thoppil, D; Lee, S; Nelson, S; Kolls, J K, AcuteAlcohol Inhibits TNF-alpha Processing in Human Monocytes by InhibitingTNF/TNF-alpha-Converting Enzyme Interactions in the Cell Membrane. 2003:p. 2923-2931.

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[0668] Equivalents

[0669] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

What is claimed is:
 1. An adzyme for enzymatically altering a substrate,the adzyme comprising: a catalytic domain that catalyzes a chemicalreaction converting said substrate to one or more products, and atargeting moiety that reversibly binds with an address site on saidsubstrate or with an address site on a second molecule that occurs infunctional proximity to the substrate, wherein said targeting moiety andsaid catalytic domain are heterologous with respect to each other, saidtargeting moiety, when provided separately, binds to the substrate, saidcatalytic domain, when provided separately, catalyzes the chemicalreaction converting said substrate to one or more products, and saidadzyme has one or more properties, with respect to the reaction withsaid substrate, of (a) a potency at least 2 times greater than thecatalytic domain or the targeting moiety alone; (b) a k_(on) of 10³M⁻¹s⁻¹ or greater; (c) a k_(cat) of 0.1 sec⁻¹ or greater; (d) a K_(D)that is at least 5 fold less than the K_(m) of the catalytic domain; (e)a k_(off) of 10⁻⁴ sec⁻¹ or greater, (f) a catalytic efficiency at least5 fold greater than the catalytic efficiency of the catalytic domainalone, (g) a K_(m) at least 5 fold less than the K_(m) of the catalyticdomain alone, and/or (h) an effective substrate concentration that is atleast 5 fold greater than the actual substrate concentration.
 2. Anadzyme for enzymatically altering a substrate, the adzyme comprising: acatalytic domain that catalyzes a chemical reaction converting saidsubstrate to one or more products, and a targeting moiety thatreversibly binds with an address site on said substrate or with anaddress site on a second molecule that occurs in functional proximity tothe substrate, wherein said substrate is an extracellular signalingmolecule, said targeting moiety and said catalytic domain areheterologous with respect to each other, said targeting moiety, whenprovided separately, binds to the substrate, said catalytic domain, whenprovided separately, catalyzes the chemical reaction converting saidsubstrate to one or more products, and said adzyme is more potent thansaid catalytic domain or targeting moiety with respect to the reactionwith said substrate.
 3. An adzyme for enzymatically altering asubstrate, the adzyme comprising a polypeptide comprising: a catalyticdomain that catalyzes a chemical reaction converting said substrate toone or more products, a targeting domain that reversibly binds with anaddress site on said substrate or with an address site on a secondmolecule that occurs in functional proximity to the substrate, and alinker joining said catalytic domain and said targeting domain, whereinsaid substrate is a receptor, said targeting moiety and said catalyticdomain are heterologous with respect to each other, said targetingdomain, when provided separately, binds to the substrate, said catalyticdomain, when provided separately, catalyzes the chemical reactionconverting said substrate to one or more products, and said adzyme ismore potent than said catalytic domain or targeting moiety with respectto the reaction with said substrate.
 4. An adzyme for enzymaticallyaltering a substrate, the adzyme comprising: a catalytic domain thatcatalyzes a chemical reaction converting said substrate to one or moreproducts, and a targeting moiety that reversibly binds with an addresssite on said substrate or with an address site on a second molecule thatoccurs in functional proximity to the substrate, wherein one or more ofsaid products is an antagonist of an activity of said substrate.
 5. Anadzyme for enzymatically altering a substrate, the adzyme comprising: acatalytic domain that cleaves at least one peptide bond of saidsubstrate to produce one or more products, and a polypeptide targetingdomain that reversibly binds with an address site on said substrate orwith an address site on a second molecule that occurs in functionalproximity to the substrate, wherein said chimeric protein construct isresistant to cleavage by the catalytic domain, said targeting moiety,when provided separately, binds to the substrate, said catalytic domain,when provided separately, cleaves at least one peptide bond of saidsubstrate to produce one or more products, and said adzyme is morepotent than said catalytic domain or targeting moiety with respect tothe reaction with said substrate.
 6. An adzyme for enzymaticallyaltering a substrate, the adzyme comprising a polypeptide comprising: acatalytic domain that catalyzes a chemical reaction converting saidsubstrate to one or more products, a targeting domain that reversiblybinds with an address site on said substrate or with an address site ona second molecule that occurs in functional proximity to the substrate,and a linker joining said catalytic domain and said targeting domain,wherein said substrate is an extracellular polypeptide signalingmolecule, said targeting moiety and said catalytic domain areheterologous with respect to each other, said targeting domain, whenprovided separately, binds to said substrate, said catalytic domain,when provided separately, catalyzes the chemical reaction convertingsaid substrate to one or more products, and said adzyme is more potentthan said catalytic domain or targeting moiety with respect to thereaction with said substrate.
 7. The adzyme of claim 1, wherein thesubstrate is endogenous to a human patient.
 8. The adzyme of claim 7,wherein the effect of the adzyme on the substrate is effective againstthe target molecule in the presence of physiological levels of anabundant human serum protein.
 9. The adzyme of claim 8, wherein theabundant human serum protein is human serum albumin.
 10. The adzyme ofclaim 2, wherein said adzyme has one or more properties, with respect tothe reaction with said substrate, of (a) a potency at least 2 timesgreater than the catalytic domain or the targeting moiety alone; (b) ak_(on) of 10³ M⁻¹s⁻¹ or greater; (c) a K_(cat) of 0.1 sec⁻¹ or greater;(d) a K_(D) that is at least 5 fold less than the K_(m) of the catalyticdomain; (e) a k_(off) of 10⁻⁴ sec⁻¹ or greater, (f) a catalyticefficiency at least 5 fold greater than the catalytic efficiency of thecatalytic domain alone, (g) a K_(m) at least 5 fold less than the K_(m)of the catalytic domain alone, and/or (h) an effective substrateconcentration that is at least 5 fold greater than the actual substrateconcentration.
 11. The adzyme of claim 10, wherein the potency of theadzyme is at least 5 times greater than the catalytic domain or thetargeting moiety alone.
 12. The adzyme of claim 10, wherein the k_(on)is 10⁶ M⁻¹s⁻¹ or greater.
 13. The adzyme of claim 10, wherein theK_(cat) is 10 sec⁻¹ or greater.
 14. The adzyme of claim 10, wherein theK_(D) is at least 50 fold lower than the K_(m) of the catalytic domain.15. The adzyme of claim 10, wherein the k_(off) is 10⁻³ s⁻¹ or greater.16. The adzyme of claim 10, wherein the catalytic efficiency is at least20 fold greater than that of the catalytic domain alone. 17 The adzymeof claim 10, wherein the K_(m) is at least 20 fold less than that of thecatalytic domain alone.
 18. The adzyme of claim 1, wherein the potencyof the adzyme is at least 5 times greater than the catalytic domain orthe targeting moiety alone.
 19. The adzyme of claim 1, wherein thek_(on) is 10⁶M⁻¹s⁻¹ or greater.
 20. The adzyme of claim 1, wherein theK_(cat) is 10 sec⁻¹ or greater.
 21. The adzyme of claim 1, wherein theK_(D) is at least 50 fold lower than the K_(m) of the catalytic domain.22. The adzyme of claim 1, wherein the k_(off) is 10⁻³ s⁻¹ or greater.23. The adzyme of claim 1, wherein the catalytic efficiency is at least20 fold greater than that of the catalytic domain alone.
 24. The adzymeof claim 1, wherein the K_(m) is at least 20 fold less than that of thecatalytic domain alone.
 25. The adzyme of claim 1, wherein said adzymeis a fusion protein.
 26. The adzyme of claim 25, wherein said fusionprotein includes a linker between said catalytic domain and saidtargeting moiety.
 27. The adzyme of claim 25, wherein said linker is anunstructured peptide.
 28. The adzyme of claim 3, wherein said linker isan unstructured peptide.
 29. The adzyme of claim 27, wherein said linkerincludes one or more repeats of Ser₄Gly or SerGly₄.
 30. The adzyme ofclaim 3, wherein said linker includes one or more repeats of Ser₄Gly orSerGly₄.
 31. The adzyme of claim 25, wherein said linker is selected toprovide steric geometry between said catalytic domain and said targetingmoiety such that said adzyme is more potent than said catalytic domainor targeting moiety with respect to the reaction with said substrate.32. The adzyme of claim 3, wherein said linker is selected to providesteric geometry between said catalytic domain and said targeting moietysuch that said adzyme is more potent than said catalytic domain ortargeting moiety with respect to the reaction with said substrate. 33.The adzyme of claim 25, wherein said linker is selected to providesteric geometry between said catalytic domain and said targeting moietysuch that said address moiety presents the substrate to the enzymaticdomain at an effective concentration at least 5 fold greater than wouldbe present in the absence of the address moiety.
 34. The adzyme of claim3, wherein said linker is selected to provide steric geometry betweensaid catalytic domain and said targeting moiety such that said addressmoiety presents the substrate to the enzymatic domain at an effectiveconcentration at least 5 fold greater than would be present in theabsence of the address moiety.
 35. The adzyme of claim 25, wherein thefusion protein is a cotranslational fusion protein encoded by arecombinant nucleic acid.
 36. The adzyme of claim 3, wherein the fusionprotein is a cotranslational fusion protein encoded by a recombinantnucleic acid.
 37. The adzyme of claim 1, wherein the substrate is abiomolecule produced by a cell.
 38. The adzyme of claim 1, wherein saidsubstrate is a polypeptide.
 39. The adzyme of claim 1, wherein thesubstrate is a polysacccaride, a nucleic acid, a lipid, or a smallmolecule.
 40. The adzyme of claim 1, wherein the substrate is adiffusible extracellular molecule.
 41. The adzyme of claim 40, whereinthe diffusible extracellular molecule is an extracellular signalingmolecule.
 42. The adzyme of claim 41, wherein the extracellularsignaling molecule is an extracellular polypeptide signaling molecule.43. The adzyme of claim 40, wherein the extracellular signaling moleculeis selected from among: interleukin-1 and TNF-alpha.
 44. The adzyme ofclaim 41, wherein the extracellular signaling molecule binds to a cellsurface receptor and triggers receptor-mediated cellular signaling. 45.The adzyme of claim 1, wherein said substrate is a receptor.
 46. Theadzyme of claim 45, wherein said substrate is a unique receptor subunitof a heteromeric receptor complex.
 47. The adzyme of claim 37, whereinthe biomolecule is a component of a biomolecular accretion.
 48. Theadzyme of claim 47, wherein the biomolecular accretion is selected fromamong: an amyloid deposit and an atherosclerotic plaque.
 49. The adzymeof claim 37, wherein the biomolecule is a biomolecule produced by apathogen.
 50. The adzyme of claim 49, wherein the pathogen is selectedfrom among, a protozoan, a fungus, a bacterium and a virus.
 51. Theadzyme of claim 37, wherein the biomolecule is a prion protein.
 52. Theadzyme of claim 1, wherein said substrate comprises a polypeptide andwherein said catalytic domain is a protease that cleaves at least onepeptide bond of the substrate.
 53. The adzyme of claim 52, wherein saidadzyme is resistant to cleavage by said catalytic domain.
 54. The adzymeof claim 3, wherein said catalytic domain is a protease that cleaves atleast one peptide bond of the substrate.
 55. The adzyme of claim 54,wherein said adzyme is resistant to cleavage by said catalytic domain.56. The adzyme of claim 52, wherein said protease is a zymogen.
 57. Theadzyme of claim 54, wherein said protease is a zymogen.
 58. The adzymeof claim 52, wherein said adzyme is purified from a cell culture in thepresence of a reversible protease inhibitor.
 59. The adzyme of claim 54,wherein said adzyme is purified from a cell culture in the presence of areversible protease inhibitor.
 60. The adzyme of claim 1, wherein saidcatalytic domain modifies one or more pendant groups of said substrate.61. The adzyme of claim 1, wherein said substrate includes a chiralatom, and said catalytic domain alters the ratio of stereoisomers. 62.The adzyme of claim 38, wherein said catalytic domain alters the levelof post-translational modification of the polypeptide substrate.
 63. Theadzyme of claim 6, wherein said catalytic domain alters the level ofpost-translational modification of the polypeptide substrate.
 64. Theadzyme of claim 62, wherein the post-translation modification isselected from the group consisting of glycosylation, phosphorylation,sulfation, fatty acid modification, alkylation, prenylation andacylation.
 65. The adzyme of claim 63, wherein the post-translationmodification is selected from the group consisting of glycosylation,phosphorylation, sulfation, fatty acid modification, alkylation,prenylation and acylation.
 66. The adzyme of claim 1, wherein thecatalytic domain is selected from the group consisting of: a protease,an esterase, an amidase, a lactamase, a cellulase, an oxidase, anoxidoreductase, a reductase, a transferase, a hydrolase, an isomerase, aligase, a lipase, a phospholipase, a phosphatase, a kinase, a sulfatase,a lysozyme, a glycosidase, a nuclease, an aldolase, a ketolase, a lyase,a cyclase, a reverse transcriptase, a hyaluronidase, an amylase, acerebrosidase and a chitinase.
 67. The adzyme of claim 3, wherein thecatalytic domain is selected from the group consisting of: a protease,an oxidase, an oxidoreductase, a reductase, a transferase, a hydrolase,an isomerase, a lipase, a phospholipase, a phosphatase, a kinase, asulfatase, a glycosidase, an aldolase, a ketolase, a lyase, a cyclase, ahyaluronidase, and a cerebrosidase.
 68. The adzyme of claim 1, whereinthe adzyme is resistant to autocatalysis.
 69. The adzyme of claim 68,wherein the adzyme is resistant to autocatalysis at an adzymeconcentration that is about equal to the concentration of adzyme in asolution to be administered to a subject.
 70. The adzyme of claim 37,wherein said adzyme alters the half-life of the biomolecule in vivo. 71.The adzyme of claim 2, wherein said adzyme alters the half-life of thesubstrate in vivo.
 72. The adzyme of claim 37, wherein said adzymealters the distribution of the biomolecule in vivo.
 73. The adzyme ofclaim 2, wherein said adzyme alters the distribution of the substrate invivo.
 74. The adzyme of claim 37, wherein said adzyme reduces abiological activity of said biomolecule.
 75. The adzyme of claim 2,wherein said adzyme reduces a biological activity of said substrate. 76.The adzyme of claim 37, wherein said biomolecule binds a plurality ofdifferent molecules in vivo, and said adzyme alters the bindingspecificity of said biomolecule.
 77. The adzyme of claim 2, wherein saidsubstrate binds a plurality of different molecules in vivo, and saidadzyme alters the binding specificity of said substrate.
 78. The adzymeof claim 37, wherein said adzyme alters the interaction of saidbiomolecule with other molecules in vivo.
 79. The adzyme of claim 2,wherein said adzyme alters the interaction of said substrate with othermolecules in vivo.
 80. The adzyme of claim 78, which alters one or moreof: a receptor-ligand interaction, a protein-protein interaction and aDNA-protein interaction.
 81. The adzyme of claim 79, which alters one ormore of: a receptor-ligand interaction, a protein-protein interactionand a DNA-protein interaction.
 82. The adzyme of claim 37, which reducesreceptor-mediated or ion channel-mediated signal transduction.
 83. Theadzyme of claim 3, which reduces receptor-mediated or ionchannel-mediated signal transduction.
 84. The adzyme of claim 37, whichalters proliferation, differentiation or viability of a cell in vivo orin vitro.
 85. The adzyme of claim 2, which alters proliferation,differentiation or viability of a cell in vivo or in vitro.
 86. Theadzyme of claim 1, wherein said product of said chemical reaction is anantagonist of said substrate.
 87. The adzyme of claim 1, wherein saidproduct of said chemical reaction has an increased biological activityrelative to said substrate.
 88. The adzyme of claim 37, which alters anintrinsic enzymatic activity of said biomolecule.
 89. The adzyme ofclaim 2, which alters an intrinsic enzymatic activity of said substrate.90. The adzyme of claim 1, wherein said substrate is a polypeptide. 91.The adzyme of claim 90., wherein said polypeptide is present inbiological fluid of an animal.
 92. The adzyme of claim 6, wherein saidextracellular polypeptide signaling molecule is present in biologicalfluid of an animal.
 93. The adzyme of claim 91, wherein said biologicalfluid is blood or lymph.
 94. The adzyme of claim 92, wherein saidbiological fluid is blood or lymph.
 95. The adzyme of claim 90, whereinsaid polypeptide substrate is a polypeptide hormone, a growth factorand/or a cytokine.
 96. The adzyme of claim 92, wherein saidextracellular polypeptide signaling molecule is a polypeptide hormone, agrowth factor and/or a cytokine.
 97. The adzyme of claim 91, whereinsaid polypeptide is selected from the group consisting of four-helixbundle factors, EGF-like factors, insulin-like factors, trefoil factorsand cysteine knot factors.
 98. The adzyme of claim 92, wherein saidextracellular polypeptide signaling molecule is selected from the groupconsisting of four-helix bundle factors, EGF-like factors, insulin-likefactors, β-trefoil factors and cysteine knot factors.
 99. The adzyme ofclaim 91, wherein said polypeptide is a pro-inflammation mediator andsaid enzyme construct reduces the pro-inflammatory activity of saidpolypeptide factor.
 100. The adzyme of claim 92, wherein saidextracellular polypeptide signaling molecule is a pro-inflammationmediator and said enzyme construct reduces the proinflammatory activityof said polypeptide factor.
 101. The adzyme of claim 91, wherein saidpolypeptide is Interleukin-1 or TNFα, and said adzyme reduces theactivity of the substrate in vivo.
 102. The adzyme of claim 92, whereinsaid extracellular polypeptide signaling molecule is Interleukin-1 orTNFα, and said adzyme reduces the activity of the substrate in vivo.103. The adzyme of claim 1, wherein the substrate is an intracellularbiomolecule.
 104. The adzyme of claim 103, wherein said adzyme furthercomprises a transcytosis moiety that promotes transcytosis of the adzymeinto the cell.
 104. The adzyme of claim 1, wherein the targeting moietycomprises a polypeptide or polypeptide complex.
 105. The adzyme of claim1, wherein said targeting moiety is a polyanionic or polycatonic bindingagent.
 106. The adzyme of claim 1, wherein said targeting moiety is anoligonucleotides, a polysaccharide or a lectin
 107. The adzyme of claim1, wherein the targeting moiety is an antibody or polypeptide(s)including an antigen binding site thereof.
 108. The adzyme of claim 107,wherein the targeting moiety is selected from the group consisting of amonoclonal antibody, an Fab and F(ab)₂, an scFv, a heavy chain variableregion and a light chain variable region.
 109. The adzyme of claim 1,wherein said substrate is receptor ligand, and said targeting moietyincludes a ligand binding domain of a cognate receptor of said ligand.110. The adzyme of claim 1, wherein said targeting moiety is anartificial protein or peptide sequence engineered to bind to saidsubstrate.
 111. The adzyme of claim 1, wherein said substrate is areceptor, and said targeting moiety is a cognate ligand of saidreceptor.
 112. The adzyme of claim 3, wherein said targeting moiety is acognate ligand of said receptor.
 113. The adzyme of claim 101, whereinthe substrate is TNFα and wherein the targeting moiety binds to TNFα.114. The adzyme of claim 102, wherein the substrate is TNFα and whereinthe targeting moiety binds to TNFα.
 115. The adzyme of claim 113,wherein the catalytic domain comprises a protease that decreases TNFαpro-apoptotic activity.
 116. The adzyme of claim 114, wherein thecatalytic domain comprises a protease that decreases TNFα pro-apoptoticactivity.
 117. The adzyme of claim 115, wherein the protease is selectedfrom among: MT1-MMP; MMP12; tryptase; MT2-MMP; elastase; MMP7;chymotrypsin; and trypsin.
 118. The adzyme of claim 116, wherein theprotease is selected from among: MT1MMP; MMP12; tryptase; MT2-MMP;elastase; MMP7; chymotrypsin; and trypsin.
 119. The adzyme of claim 113,wherein the targeting moiety is selected from among, a soluble portionof a TNFα receptor and a single chain antibody that binds to TNFα. 120.The adzyme of claim 114, wherein the targeting moiety is selected fromamong, a soluble portion of a TNFα receptor and a single chain antibodythat binds to TNFα.
 121. The adzyme of claim 113, wherein the targetingmoiety is an sp55 portion of TNFR1.
 122. The adzyme of claim 114,wherein the targeting moiety is an sp55 portion of TNFR1.
 123. Theadzyme of claim 101, wherein the substrate is IL[-1 and wherein thetargeting moiety binds to IL-1.
 124. The adzyme of claim 102, whereinthe substrate is IL-1 and wherein the targeting moiety binds to IL-1.125. The adzyme of claim 123, wherein the catalytic domain comprises aprotease that decreases an IL-1 bioactivity.
 126. The adzyme of claim124, wherein the catalytic domain comprises a protease that decreases anIL-1 bioactivity.
 127. An adzyme preparation for therapeutic use in ahuman patient, the preparation comprising an adzyme of claim
 1. 128. Theadzyme preparation of claim 127, further comprising a pharmaceuticallyeffective carrier.
 129. The adzyme preparation of claim 127, wherein theadzyme preparation is formulated such that autocatalytic modification ofthe adzyme is inhibited.
 130. The adzyme preparation of claim 129,wherein the adzyme comprises a catalytic domain that is a protease. 131.The adzyme preparation of claim 130, further comprising a reversibleinhibitor of said protease.
 132. The adzyme preparation of claim 131,wherein the reversible inhibitor is safe for administration to a humanpatient.
 133. The adzyme preparation of claim 127, wherein said adzymepreparation is substantially pyrogen free.
 134. The adzyme preparationof claim 127, wherein said adzyme preparation is packaged withinstructions for administration to a patient.
 135. A method of making amedicament for use in treating a disorder that is associated with anactivity of the substrate of an adzyme of claim 1, the method comprisingformulating the adzyme for administration to a human patient.
 136. Amethod of making a medicament for use in treating an inflammatory orallergic disorder, the method comprising formulating an adzyme of claim1 for administration to a human patient in need thereof, wherein thesubstrate of the adzyme is an inflammatory cytokine.
 137. A method oftreating a disorder that is associated with an activity of the substrateof an adzyme of claim 1, the method comprising administering atherapeutically effective dose of the adzyme to a human patient in needthereof.
 138. A method of treating an inflammatory of allergic disorder,the method comprising administering a therapeutically effective dose ofan adzyme to a human patient in need thereof, wherein the substrate ofthe adzyme is an inflammatory cytokine.
 139. A nucleic acid comprising acoding sequence for the adzyme of claim
 3. 140. A nucleic acid encodinga coding sequence for the adzyme of claim
 25. 141. An expression vectorcomprising the nucleic acid of claim 139, wherein the expression vectordirects expression of the adzyme in a suitable host cell.
 142. Anexpression vector comprising the nucleic acid of claim 140, wherein theexpression vector directs expression of the adzyme in a suitable hostcell.
 143. A cell comprising the expression vector of claim
 141. 144. Acell comprising the expression vector of claim
 142. 145. A cellcomprising a first nucleic acid comprising a first coding sequence and asecond nucleic acid comprising a second coding sequence, wherein thefirst coding sequence encodes a first fusion protein comprising animmunoglobulin heavy chain and a catalytic domain, and wherein thesecond coding sequence encodes a second fusion protein comprising animmunoglobulin heavy chain and a targeting domain.
 146. The cell ofclaim 145, wherein, in appropriate culture conditions, the cell secretesan adzyme comprising an Fc fusion protein construct that is a dimer ofthe first fusion protein and the second fusion protein.
 147. A methodfor manufacturing an adzyme, the method comprising a) culturing a cellof claim 143 in conditions that cause the cell to produce the adzymeencoded by the expression vector; and b) purifying the adzyme tosubstantial purity.
 148. A method for manufacturing an adzyme, themethod comprising a) culturing a cell of claim 144 in conditions thatcause the cell to produce the adzyme encoded by the expression vector;and b) purifying the adzyme to substantial purity.
 149. A method formanufacturing an adzyme, the method comprising a) culturing a cell ofclaim 146 in conditions that cause the cell to produce the adzyme; andb) purifying the adzyme to substantial purity.
 150. The method of claim147, wherein the purifying the adzyme to substantial purity includes theuse of a reversible inhibitor that inhibits autocatalytic activity ofthe catalytic domain.
 151. The method of claim 147, wherein thecatalytic domain of the adzyme is a protease domain, and whereinpurifying the adzyme to substantial purity includes the use of areversible protease inhibitor that inhibits the protease activity of thecatalytic domain.
 152. A method of designing and constructing aneffective adzyme, the method comprising: a) selecting a substrate thatis a known target for a therapeutically effective binding agent; b)testing a plurality of catalytic domains for effectiveness in reducingan activity of the substrate to obtain a set of one or more candidatecatalytic domains that are effective in reducing an activity of thesubstrate; c) testing a plurality of binding moieties for effectivenessin binding to the substrate to obtain a set of one or more candidatetargeting moieties that are effective in binding to the substrate; d)constructing and producing a plurality of adzymes comprising one or moreof the candidate catalytic domains and one or more of the candidatetargeting moieties, wherein the one or more catalytic domains and theone or more candidate targeting moieties are associated in at least twodifferent geometric conformations, e) testing the plurality of adzymesfor effectiveness in reducing an activity of the substrate to obtain aset of one or more candidate adzymes, wherein an adzyme that iseffective for reducing an activity of the substrate is an effectiveadzyme.
 153. The method of claim 152, further comprising testing theefficacy of the adzyme in an organism.
 154. The method of claim 152,further comprising modifying an effective adzyme to improve one or moreof the following properties: a) reduce the amount of autocatalysis; b)increase the potency of the adzyme; c) increase the specificity of theadzyme; d) improve the balance of the potency and specificity of theadzyme; e) increase the serum half-life of the adzyme; f) decrease theinteractions between the adzyme and one or more abundant serum proteins.155. A method of operating a therapeutic adzyme business, the methodcomprising: a) designing an adzyme according to the method of claim 152;b) testing the adzyme for safety and effectiveness in humans; c)arranging for distribution and marketing of the adzyme.